In this study, we investigated the combined treatment of 5‐fluorouracil (5‐FU) and Anatolian propolis extract (PE) on colorectal cancer (CRC)using in vitro and in vivo studies. We exposed luciferase‐transfected (Lovo‐Luc CRC) cells and healthy colon cells (CCD‐18Co) to varying concentrations of 5‐FU and PE to assess their genotoxic, apoptotic, and cytotoxic effects, as well as their intracellular reactive oxygen species (iROS) levels. We also developed a xenograft model in nude mice and evaluated the anti‐tumor effects of PE and 5‐FU using various methods. Our findings showed that the combination of PE and 5‐FU had selectivity against cancer cells, particularly at higher doses, and enhanced the anti‐tumor effectiveness of 5‐FU against colon CRC. The results suggest that PE can reduce side effects and increase the effectiveness of 5‐FU through iROS generation in a dose‐dependent manner.