Aim <p>Esophagectomy may alter gut microbiota, a key regulator of antitumor immunity and immune checkpoint inhibitor (ICI) efficacy; however, its impact in patients with esophageal squamous cell carcinoma (ESCC) remains unclear. This study evaluated the association between the tumor immune microenvironment (TIME) and gut microbiota and also investigated gut microbiota composition in relation to nivolumab response in patients with ESCC.</p> Methods <p>Two cohorts were analyzed. In Cohort 1 (n = 18), fecal samples collected before and three months after esophagectomy were analyzed alongside TIME profiling, and patients were classified as hot or cold tumors based on CD8⁺ T-cell infiltration and PD-L1 expression. In Cohort 2 (n = 35), the relationship between baseline gut microbiota and nivolumab response was assessed using a retrospective analysis of a prospectively collected multicenter cohort.</p> Results <p>Patients with hot tumors exhibited higher microbial alpha diversity than those with cold tumors. Before esophagectomy, cold tumors showed enrichment of Streptococcus, Veillonella, Haemophilus, and Gemella, whereas hot tumors were enriched in Flavonifractor and Phascolarctobacterium; these differences disappeared postoperatively, indicating surgery-associated compositional shifts. In Cohort 2, higher alpha diversity was associated with favorable nivolumab response, while patients with Streptococcus- or Veillonella-enriched microbiota more frequently had residual tumors and poor outcomes.</p> Conclusion <p>Gut microbiota diversity was associated with the tumor immune microenvironment and nivolumab response in patients with esophageal cancer. Esophagectomy was also associated with alterations in gut microbial composition.</p> Graphical abstract <p></p>