Purpose <p>Preoperative lymphadenopathy in colorectal cancer may represent nodal metastasis or reactive change. We aimed to compare gut microbiota profiles between reactive and metastatic lymphadenopathy and to explore microbial features associated with nonmetastatic lymph node enlargement.</p> Methods <p>We conducted a retrospective observational study of colorectal cancer patients with radiological lymphadenopathy who underwent resection at Shiga University of Medical Science Hospital between 2018 and 2021. For the primary analysis, we included patients with radiological lymphadenopathy on preoperative CT. An additional analysis restricted to right-sided colon cancer compared no lymphadenopathy without pathological lymph node metastasis (NN), lymphadenopathy without pathological lymph node metastasis (PN), and lymphadenopathy with pathological lymph node metastasis (PP).</p> Results <p>Pathological nodal metastasis was identified in 34 patients, whereas 29 had reactive lymphadenopathy. Fecal samples showed higher alpha diversity than tumor tissues. In left-sided colorectal cancer, no notable bacterial taxa exceeded the predefined LDA threshold. In right-sided colon cancer, Proteobacteria were enriched in metastatic cases, whereas Firmicutes were more abundant in reactive lymphadenopathy. Predicted pathway analysis suggested distinct metabolic profiles between the two groups. In the additional right-sided analysis, alpha and beta diversity did not significantly differ among NN, PN, and PP, although taxon-level differences were observed between NN and PN.</p> Conclusion <p>Gut microbiota profiles differed between reactive and metastatic lymphadenopathy in colorectal cancer, with more distinct findings in right-sided tumors. These findings suggest an association between microbial patterns and reactive lymphadenopathy.</p>