TP53 mutations drive colorectal cancer development, with missense mutations frequently leading to accumulation of abnormal TP53 protein. TP53 alterations have been associated with poor prognosis and chemotherapy resistance, but data remain controversial. Here, we examined the predictive utility of TP53 overexpression in the context of current adjuvant treatment practice for patients with stage III colorectal cancer. A prospective cohort of 264 stage III patients was tested for association of TP53 expression with 5-year disease-free survival, grouped by adjuvant treatment. Findings were validated in an independent retrospective cohort of 274 stage III patients. Overexpression of TP53 protein (TP53+) was found in 53% and 52% of cases from the prospective and retrospective cohorts, respectively. Among patients receiving adjuvant chemotherapy, TP53+ status was associated with shorter disease-free survival (p ≤ 0.026 for both cohorts), while no difference in outcomes between TP53+ and TP53- cases was observed for patients treated with surgery alone. Considering patients with TP53- tumors, those receiving adjuvant treatment had better outcomes compared with those treated with surgery alone (p ≤ 0.018 for both cohorts), while no treatment benefit was apparent for patients with TP53+ tumors. Combined cohort-stratified analysis adjusted for clinicopathological variables and DNA mismatch repair status confirmed a significant interaction between TP53 expression and adjuvant treatment for disease-free survival (p = 0.030). For the combined cohort, the multivariate hazard ratio for TP53 overexpression among patients receiving adjuvant chemotherapy was 2.03 (95% confidence interval 1.41-2.95, p < 0.001), while the hazard ratio for adjuvant treatment among patients with TP53- tumors was 0.42 (95% confidence interval 0.24-0.71, p = 0.001). Findings were maintained irrespective of tumor location or when restricted to mismatch repair-proficient tumors. Our data suggest that adjuvant chemotherapy benefit in stage III colorectal cancer is restricted to cases with low-level TP53 protein expression. Identifying TP53+ tumors could highlight patients that may benefit from more aggressive treatment or follow-up.