Colorectal cancer (CRC) is one of the most common cancers worldwide. Although chemotherapy is used as a palliative treatment, ultimately, nearly all patients develop drug resistance. Therefore, the cell-inherent DNA repair pathway must reverse the DNA-damaging effect of cytotoxic drugs that mediates therapeutic resistance to chemotherapy. RAD18, a DNA damage-activated E3 ubiquitin ligase, is known to play a critical role in DNA damage repair in cancer cells. Here, we show that RAD18 is highly expressed in human 5-fluorouracil (5-FU)-resistant cancer cells after 5-FU treatment. In addition, RAD18 increases in CRC cells could induce DNA damage repair, suggesting that RAD18 might be a possible target for overcoming drug resistance. Moreover, the expression of tumor suppressor microRNA-145 (miR-145) was negatively correlated with RAD18 expression in CRC tissues of 140 patients. Using luciferase reporters carrying the 3'-untranslated region of RAD18 combined with Western blotting, we identified RAD18 as a direct target of miR-145. Also of interest, suppression of RAD18 by miR-145 enhanced DNA damage in CRC cells after 5-FU treatment. Finally, the 5-FU-resistant cancer cells could be selectively ablated by treatment with miR-145. Taken together, these results suggest that miR-145 can act as an RAD18 inhibitor and contribute as an important factor in reversing drug resistance after chemotherapy.