CDK2-associated cullin domain 1 (CAC1) is as a novel cell cycle regulator widely expressed in colorectal cancer (CRC). However, its expression and function in drug resistant CRC cells remains elusive. Therefore, the present study aimed to assess the biochemical function and relevance of CAC1 in drug resistant CRC cells, and detect the potential mechanism. For this purpose, a total of 83 CRC cases were collected for the immunohistochemical analysis of CAC1 expression. Functional studies (stable transfection, flow cytometry, colony formation, and invasion and migration assays) were performed in SW480, LoVo and their corresponding 5-FU resistant cells. In addition, a nude mice xenograft model was established for further observation in vivo. In the present study, CAC1 protein expression was higher in CRC tissues than that in normal tissues (P<0.05). Furthermore, CAC1 protein expression was higher in SW480/5-FU cells than in SW480 cells. CAC1 knockdown arrested 5-FU resistant cells at the G1/S phase and increased the sensitivity of 5-FU resistant cells to 5-FU by inducing apoptosis. In addition, CAC1 reduced the invasive and migration ability of SW480/5-FU and LoVo/5-FU cells in vitro, and reduced their tumorigenicity and metastatic ability in vivo. Finally, CAC1 knockdown resulted in decreased P-glycoprotein and MRP-1 protein expression. Based on these results, it can be concluded that CAC1 plays an important role in the occurrence and promotion of drug resistance in CRC. Therefore, the knockdown of CAC1 may be considered as a new strategy for the development of CRC drug resistance treatments in the future.