Background and Aims Mutations in the mutL homolog 1 (MLH1) gene can lead to inactivation of the DNA mismatch repair (MMR) system, increasing the risk of colorectal cancer. Additionally, growing evidence suggests that alterations in the composition and function of the intestinal microbiota are closely associated with the occurrence and development of colorectal cancer. However, the relationship between MLH1 protein expression and the intestinal microbiota remains unclear. Therefore, this study aimed to explore the potential relationship between them by analyzing the differences in the microbial composition of tumor tissues between patients with proximal sporadic colon cancer (SCC) and different MLH1 protein phenotypes in Northeast China.Methods Tumor tissue samples and clinical data were collected from 407 patients with proximal SCC treated in Harbin First Hospital and Heilongjiang Provincial Hospital between 2020 and 2021. Immunohistochemistry was used to screen for cases with MLH1 protein deficiency (deficiency group) and intact MLH1 protein (control group). Microbial DNA extracted from the intestinal tumor tissues was analyzed using 16S rRNA gene sequencing technology for bioinformatics analysis. The relationship between clinicopathologic features and the diversity of specific taxa and microbial diversity was analyzed.Results A total of 20 cases were screened in the deficiency group, and 18 cases were screened in the control group. Preliminary analysis of clinical data showed that the larger the tumor, the higher the risk of MLH1 protein deficiency (P<0.05). The α-diversity of the microbial community within tumor tissues under different MLH1 statuses showed no statistically significant differences except for the Shannon index (P=0.042). Other diversity indices had no significant difference (all P>0.05). The β-diversity analysis of microbial communities at the phylum level showed no significant differences between the two groups (P=0.076). At the genus level, β-diversity analysis showed that the differences between the two groups were greater than those within the groups (P=0.04). A comparison of the abundance of bacterial genera revealed that the abundance of the genus Coprococcus spp may promote MLH1 protein deficiency (adjusted P<0.01). However, there were no significant differences in the Shannon diversity index between various clinicopathologic variables or key species with different abundances (all P>0.05).Conclusion The MLH1 protein phenotype of proximal SCC patients is closely related to the composition and diversity of the intestinal microbiota. Moreover, Coprococcus spp was identified as a potential key species associated with MLH1 protein loss in this population, providing a new perspective for future research, prevention, and treatment of this disease.