Colorectal cancer (CRC) accounts for 10–15% of all cancers and is the second leading cause of cancer-related deaths. The study was designed to investigate the effects of insulin on 5-fluorouracil (5-FU) anti-tumor action in human colon cancer SW-480 cell line. SW-480 cancer cell line was cultured in vitro according to the routine cell culture protocols using the specific culture media and reagents. Initially, the effect of serially double diluted concentrations of insulin and 5-FU was investigated separately on the SW-480 cell line for periods of 12 and 24 hours in five replicates of a microtiter plate under complete sterile conditions. Two concentrations of insulin, 0.5U/L and 1U/L had been chosen to be combined with different concentrations of 5-FU in the study experiments. The SW-480 cell line was pre-exposed to either insulin concentrations for periods of incubation (0h, 3h, 6h, 9h, 12h, 24h) followed by treatment with 5-FU for 12h and 24h for both insulin concentrations. Crystal violet cytotoxicity assay was used to investigate the effects of insulin, 5-FU and their combination on SW-480 cell line. The results of these experiments were studied as viability%, inhibition%, 50% of maximal inhibition and the 50% inhibitory concentration (IC50) for cell growth. Gas chromatography analysis was used to determine the residual concentration of 5-FU in the culture medium of SW-480 cell line pretreated with insulin. The glucose concentration in culture medium was determined at the end of the incubation periods as indicator of cell viability. The SW-480 cells was stained with hematoxiline and eosin to compare between the effect of 5-FU with and without insulin. The results showed that different concentrations of insulin produced nonsignificant changes (P>0.05) in the growth of SW-480 cell line even after prolongation of its incubation period. low concentrations of 5-FU (1.25 μg/ml and 2.5 μg/ml) produced non-significant (P>0.05) decrease in the viability percent of SW-480 cell line after 6h, 12h and 24h of incubation, whereas the concentrations above 2.5μg/ml produced proportional significant (P<0.05) decrease in the viability percent of the cells . On the other hand, the significant decrease in the viability percent for these two concentrations with prolongation of incubation period , indicated that 5-FU at higher concentrations decreases the cancer growth in a dose and time dependent manner. The effects of concomitant use of insulin and 5-FU produced non-significantm changes (P>0.05) in the viability percent of SW-480 cell line as compared with the same concentrations of 5-FU without insulin. Obviously prolongation of the incubation period resulted in an augmented effect of 5-FU significantly (P<0.05), starting from 5 μg/ml up to 80 μg/ml of concentrations. The increment in the concentration of insulin from 0.5 to 1U/L produced non-significant (P>0.05) changes in the viability percent of SW-480 cells for different concentrations of 5- FU. Pre-treatment with insulin enhanced significantly (P<0.05) the cytotoxic action of the low concentrations as well as the high concentrations of 5-FU on the proliferation of SW-480 cell line after exposure for 3h,6h, 9h, 12h and 24h. The results also showed that prolongation the time of exposure to insulin from 3h- 9h at both periods of incubation produced gradual significant (P<0.05) increment in the effect of different concentrations of 5-FU on SW-480 cell line. However, if the exposure time to insulin was increased to 12 and 24h, the viability percent did not change significantly (P>0.05) while, increasing the period of 5FU incubation from 12h to 24h produced significant differences (P<0.05) for its effects. On the contrary, increasing insulin concentration from 0.5U/L to 1U/L did not enhance the action of 5-FU any more. These results indicate that the enhancement of insulin on the action of 5-FU was time rather than dose dependent. The growth of SW-480 cell line was inhibited significantly (P<0.05) by 5-FU concentrations above 2.5μg/ml and significant (P<0.05) increase in the 50% of the maximal inhibition with non-significant (P>0.05) increase in IC50 according to the period of incubation. The 50% of maximal inhibition of 5-FU pre-treated with insulin increased significantly (P<0.05) with the increasing in the xposure time to insulin except for 3h group. Enhancement of 5-FU action by insulin resulted in decreasing the IC50 of 5-FU especially for the 9h, 12h and 24h groups. However, the differences among these groups were non-significant (P>0.05). Gas Chromatographic analysis revealed significant differences (P<0.05) between the residual concentrations of 5-FU in the culture medium of SW-480 cell line well replicates pre-exposed with insulin in comparison with cell line well replicates treated with 5-FU only. This indicates that insulin enhance the uptake and transport of 5-FU by the SW-480 cell line. There was significant decrease (P<0.05) in glucose value of the culture medium for all the concentrations of insulin, While there was significant increase (P<0.05) in glucose value for 5-FU concentrations starting from 5μl/ml comparing with the control groups. Such result showed that there was an inverse correlation between the activity of 5-FU and the glucose level on SW-480 cell line, so glucose determination may be a valuable predictor of an in vitro anticancer drug activity. Histological examination showed presence of large number of apoptotic and pre-apoptotic cells, and a malignant cells with karyolytic nuclei, which were more prevalent in SW-480 cell line treated with 5-FU preceeded with insulin than with 5-FU alone. These results confirmed the enhancement of anti-tumor action of 5-FU by insulin. In conclusion, the pre-treatment of SW-480 cell line with neutral insulin for nine hours will enhance the in vitro cytotoxic action of 5-FU significantly in comparison to the use of 5-FU alone