RNF43 is a tumor suppressor for various cancers and is considered to drive carcinogenesis when mutated. However, the correlation between RNF43 mutation and colorectal cancer (CRC) immunotherapy remains unreported. We evaluated the role of RNF43 using publicly available data from the Cancer Genome Atlas (TCGA) and Memorial Sloan Kettering Cancer Center (MSKCC). In addition, further analysis was performed on an internal validation cohort (hcohort). The mutant profiles of RNF43 were analyzed in 873 Chinese CRC patients. The relationship between clinical pathologic features and RNF43 were analyzed using the two-sided chi-squared test or the Fisher exact test. Clinicopathologic characteristics were associated with overall survival using Cox regression and the Kaplan-Meier method. We found that RNF43 mutation was significantly associated with high TMB and high MSI score (all p-values < 0.05) in the MSKCC cohort. Additionally, RNF43 mutation was found to be enriched in MSI instability. Kaplan-Meier survival analysis revealed that patients with RNF43 mutation had better OS compared to RNF43 wild-type (not reached vs. 13 months, HR, 0.12; 95% CI 0.03 to 0.49; P = 0.0034). However, no association was observed between RNF43 and OS in the TCGA cohort (HR, 1.83; 95% CI 0.66 to 5.07; P = 0.2479). Our CRC hcohort confirmed the significance of RNF43 mutation in predicting better clinical outcomes, including ORR (45% vs. 21%, P = 0.0468). RNF43 mutation correlated with a high tumor mutation burden (P < 0.001). The mutation frequency of RNF43 in CRC patients was 8.4% (73/873); RNF43 G659Vfs*41 was found to be the most frequent mutation site. In patients with RNF43 mutations, TP53, KRAS, and TGFBR2 were genes with a high frequency of mutations. Compared with RNF43 wild-type patients, those with RNF43 mutations had a higher TMB score and a greater proportion of MSI-H, but no difference in PD-L1 expression. Moreover, the content of immune-related B cells, CD8+ T cells, neutrophils, and dendritic cells was higher in the RNF43 mutant group than in the wild-type group. Our results suggest that RNF43 mutation may correlate with better OS in CRC patients receiving PD-1/PD-L1 inhibitors. The exact mechanisms underlying RNF43 require further investigation.AJCR Copyright © 2023.