We assessed the role of nutritional factors in conjunction with pathological and clinical characteristics of colorectal tumors with microsatellite instability. Data from 1,135 patients diagnosed with colorectal cancer were analyzed, employing immunohistochemistry for mismatch repair protein detection and polymerase chain reaction-capillary electrophoresis for microsatellite instability gene detection. To explore potential correlations with microsatellite instability status, we evaluated patients’ dietary habits and micronutrient levels, particularly folate and selenium. We discovered that 10.84% of the patients had mismatch repair protein deletions, with MLH1 and PMS2 double deletions being the most prevalent. Notable differences were identified in serum carcinoembryonic antigen levels, tumor location, tumor-node-metastasis stage, tumor differentiation, mucous composition, gross type, size, nodules, nerve invasion, and lymph node ratio between deficient mismatch repair and proficient mismatch repair patients. The deficient mismatch repair group had lower carcinoembryonic antigen levels, a higher incidence of right-sided colon cancer, earlier tumor-node-metastasis stage, poorer differentiation, more mucinous components, larger tumors, and fewer nodules and nerve invasions. High concordance (98.59%) was observed between immunohistochemistry and polymerase chain reaction-capillary electrophoresis techniques in microsatellite instability detection, underscoring the reliability of these methods. Integrating the nutritional parameters with conventional clinical assessments offers a nuanced understanding of the heterogeneity of colorectal cancer, with significant implications for diagnosis and treatment strategies.