Increasing evidence indicates that nuclear factor, erythroid 2-like 3 (Nrf3) is connected with tumorigenesis. However, the relationship between Nrf3 and tumor drug resistance remains elusive. In this study, we investigated the effect and mechanism of action by which Nrf3 regulated the sensitivity of colon cancer cells to 5-fluorouracil (5-FU). We found Nrf3 was significantly increased in colon cancer tissues. Furthermore, we observed that Nrf3 knockdown and overexpression can significantly affect the sensitivity of colon cancer cells to 5-FU in vitro and in vivo. Moreover, Nrf3 promoted the expression of RELA, P-RELA, and BCL-2. Inhibition of NF-B partly reversed the effects of Nrf3 overexpression, resulting in the resistance of colon cancer cells to 5-FU. Overall, the study revealed that Nrf3 was connected to the sensitivity of colon cancer cells to 5-FU, and its possible mechanism was related to the NF-B signaling pathway, which provided a new target for overcoming the resistance of colon cancer cells to 5-FU.