Chemoresistance often occurs during 5-fluorouracil (5-Fu) treatment of colorectal cancer (CRC). It is significant to explore the potential strategies to sensitize colorectal cancer cells to 5-Fu treatment. We studied the sensitization of Nephroblastoma overexpressed protein (NOV) on 5-Fu treatment. NOV was overexpressed and knocked down in HT115 and RKO cells respectively. Cell proliferation experiments and related mechanism studies by RT-qPCR and Western blot were performed Subsequently. Nude mouse xenograft model was established to test the inhibitory effect of 5-FU on CRC cells in vivo. In this study, we found that NOV mRNA expression was significantly lower in tumor tissues than that in the normal tissues (P < 0.05). The cell proliferation was reduced in the HT115-NOVexp groups (P < 0.05) and increased in the RKO-NOVkd groups (P < 0.05) than that in the control groups and NC groups. The RT-PCR and Western Blot results showed that NOV inhibited the expression of activator protein (AP)-1 (P < 0.05) and promoted the expression of Caspase-8/3 (P < 0.05) in CRC cells in vitro. NOV also improved the inhibitory effect of 5-Fu on inhibiting colorectal cancer proliferation in a tumor cell xenotransplantation nude mouse model. NOV inhibited the expression of AP-1 and JUK and promoted the expression of Caspase-8/3 in cancer tissues in a tumor cell xenotransplantation nude mouse model. In summary, NOV can sensitize CRC cells towards 5-Fu-mediated inhibitory effect on cell proliferation and its sensitization may be achieved by the JNK/AP-1/Caspase-8/Caspase-3 pathway.