Colorectal cancer (CRC) is the second‑leading cause of cancer-related mortality worldwide. It is frequently characterized by chemotherapy resistance，which is a predominant factor contributing to unfavorable patient prognosis. B7-H3 is a novel tumor marker and a potential immunotherapy target. High B7-H3 expression in colorectal cancer is associated with adverse prognosis. In this study, we noted increased B7-H3 expression in colorectal cancer tumor tissues. Both in vivo and in vitro experiments demonstrated that increased B7-H3 expression promotes resistance to chemotherapy in CRC. Furthermore, our findings suggest that B7-H3 mediates CRC resistance by modulating CYP1B1 expression. Mechanistic investigations indicated that B7-H3 inhibited the ubiquitination of CYP1B1, stabilized its expression，and consequently enhanced chemotherapeutic resistance in CRC. In summary, our results underscore the significance of the B7-H3-CYP1B1 interaction as a crucial therapeutic target for overcoming chemotherapy resistance in CRC.