Abstract: Background and Objective: Colorectal cancer (CRC) is the most influential cause of cancer death worldwide. Despite its effectiveness in CRC therapy, its clinical applications are restricted because of its short half-life, resistance and severe side effects. The present study was directed to formulate nanoparticles of 5-Fluorouracil (5-FU) in the presence of Simvastatin (SMV) in an attempt to enhance the therapeutic efficacy of 5-FU. Materials and Methods: Formulation of Solid Lipid Nanoparticles (SLN) has been performed and cytotoxic activity was tested in human colorectal cancer cell line (HCT-116) the IC50 was investigated to evaluate the cytotoxicity, apoptosis induction, cell cycle distribution and the intercellular Reactive Oxygen Species (ROS) after treatment with SLN 5-FU and/or SMV compared with raw drug. Results: The particles size was 107-117 nM and stability of formula between -5.53 and -14 mV, Entrapment Efficiency was 80-97.5% for 5-FU and SMV, respectively. Raw 5-FU had IC50 12.69 μM while cells treated with 5-FU SLN, IC50 dropped significantly and addition of 5 μM SMV SLN, IC50 was significantly reduced. Also, treatment with SLN 5-FU alone and/or SMV significantly accumulated the cells in sub-G1 and dramatically increased the percentage of late apoptotic cells significantly in comparison to raw 5-FU. Moreover, SMV SLN with 5-FU had increased intracellular ROS accumulation. Conclusion: The SLN formulation for both 5-FU and SMV showed a significant cytotoxic potentiating effect against the growth of HCT-116 cells.