BACKGROUND: Stage III colon cancer (CC) presents a critical therapeutic challenge due to its high recurrence risk. Identifying robust prognostic biomarkers to guide adjuvant therapy decisions is urgently needed in clinical practice. Tertiary lymphoid structures (TLSs), as immune aggregates within the tumor microenvironment, have emerged as potential indicators of immunological activity and treatment response. The objective of this study is to evaluate the role of TLSs in stage III CC, focusing on their potential as prognostic markers and their influence on patient outcomes, particularly in relation to chemotherapy response. METHODS: This retrospective cohort study enrolled 613 patients with pathologically confirmed stage III CC from two cohorts: 374 from Harbin Medical University and 239 from The Cancer Genome Atlas Colon Adenocarcinoma (TCGA-COAD) external validation cohort. Overall survival (OS) was the primary outcome, with a median follow-up period of 62 months. TLSs were assessed via immunohistochemistry and categorized by density and location [intratumoral (T score), peritumoral (P score)]. Prognostic significance was evaluated using multivariate Cox regression. A murine model was used to assess the immunomodulatory effects of folinic acid, oxaliplatin, and 5-fluorouracil (FOLFOX) chemotherapy on TLS formation. RESULTS: TLSs were present in 54.0% and 50.2% of patients in Cohorts 1 and 2, respectively. TLSs enriched with CD8 T cells and CD20 B cells were associated with improved OS. Multivariate analysis identified TLS presence as an independent predictor of better survival [hazard ratio (HR) =0.256, 95% confidence interval (CI): 0.093-0.707; P=0.009]. Higher intratumoral TLS density (T score) correlated with lower mortality risk (T2 T0: HR =0.173, P=0.003), whereas higher peritumoral TLS density (P3) predicted worse prognosis (HR =5.887, P=0.04). experiments confirmed that FOLFOX treatment enhanced TLS formation and increased infiltration of immune cells including B cells, CD4/CD8 T cells, and dendritic cells. CONCLUSIONS: TLSs serve as a reliable, independent prognostic biomarker in stage III CC. Their spatial distribution carries distinct prognostic implications, and FOLFOX-induced TLS formation suggests a dual role in cytotoxicity and immune activation. Incorporating TLS assessment into clinical workflows may improve risk stratification and guide personalized treatment, especially in designing immunochemotherapy strategies.