Background: Clinical benefit of adjuvant chemotherapy (AC) is still controversial in locally advanced rectal cancer (LARC) after neoadjuvant chemoradiation therapy (CRT) followed by total mesorectal excision (TME). We aim to explore the role of adjuvant chemotherapy with fluoropyrimidine for ypT0-3N0 patients. Methods: Patients with ypT0-3N0 rectal cancer after neoadjuvant CRT and TME were included using retrospective cohort of Yonsei Cancer Center. Patients were categorized according to receipt of adjuvant chemotherapy (AC vs. no AC). Disease free survival (DFS) and overall survival (OS) between treatment groups were compared using all patients’ cohort (APC) and propensity score-matched patients’ cohort (PSMPC). Results: total of 339 patients were evaluated. Of all, 87 patients (25.7%) did not receive AC. There was no difference in DFS between two groups [hazard ratio (HR) = 1.079, p-value = 0.782 in APC; HR = 1.22, p-value in PSMPC]. Also there was no difference in OS between two groups (HR = 1.140, p-value 0.717 in APC; HR = 1.366, p-value 0.472 in PSMPC). Advanced T stage and positive resection margin were associated with inferior DFS and OS by multivariate analysis. In subgroup analysis by baseline characteristics, we could not find any group with benefit of adjuvant chemotherapy. Conclusions: AC did not improve DFS and OS of patients with ypT0-3N0 rectal cancer after neoadjuvant CRT followed by TME. The role of AC in LARC with ypT0-3N0 after preoperative CRT should be evaluated in prospective randomized trials with larger sample size.