Background <p>Microsatellite stability influences the prognosis of patients with colorectal cancer (CRC). However, there are few studies on the relationship between microsatellite stability and lymph node metastasis (LNM) in CRC.</p> Objective <p>This study aims to elucidate the relationship between microsatellite stability and LNM in CRC and to investigate potential underlying mechanisms.</p> Methods <p>A retrospective analysis was performed on a cohort of 309 CRC patients, who were categorized into microsatellite instability (MSI) and microsatellite stability (MSS) groups based on their microsatellite status. Clinical and pathological indicators were collected, and differences between the two groups were assessed. The tertiary lymphoid structures (TLS) in both groups were examined using immunohistochemistry and immunofluorescence to compare differences in density, maturity, and the ratio of CD8+T cells. Establishing the relationship between microsatellite stability, TLS characteristics, and lymph node metastasis.</p> Results <p>The TNM staging for patients in the MSI group was significantly earlier compared to those in the MSS group. Subsequent analysis of pathological indicators demonstrated that the MSI group exhibited a significantly lower incidence of lymph node metastases (31.4% vs. 47%, P=0.005), while no statistically significant differences were observed in other pathological indicators (P&gt;0.05). Examination of CRC tissue sections revealed that the MSI group possessed a greater number and maturity of tertiary lymphoid structures, as well as a higher proportion of CD8+T cells.</p> Conclusion <p>MSI may decrease the incidence of LNM in CRC, potentially as a result of the activation of local anti-tumor immune responses facilitated by MSI.</p>