Background: Neoadjuvant chemotherapy followed by surgery (NAC-S) is the standard therapy for locally advanced esophageal squamous cell cancer (ESCC) in Japan. The aim of this phase II trial was to assess the efficacy and safety of the addition of adjuvant S-1 after R0 resection in patients treated with NAC-S. Methods: Key eligibility criteria were as follows: ESCC of clinical stage IB-III (without T4 disease); aged 20 to 75 years; ECOG performance status 0 or 1; and performed neoadjuvant chemotherapy (5-FU + cisplatin). All patients registered before surgery. Patients received adjuvant therapy with 4 cycles of S-1 (80 mg/m²/day) that is administered orally for 4 weeks of a 6-weeks cycle. The primary endpoint was three-year relapse free survival (RFS). Results: A total of 52 patients were enrolled between January 2016 and January 2019. Two patients (one with small cell carcinoma and the other with synchronous malignancy) were excluded from analysis. Five patients were diagnosed as R1 or R2. Seven patients did not receive adjuvant S-1 due to adverse events of surgery in 5 patients, refusal to adjuvant S-1 in a patient, and forgot to start in a patient. Thirty-eight patients received adjuvant S-1, with 32 patients completing 4 cycles. The median relative dose intensity of adjuvant S-1 was 85.8%. Median follow-up time among survivors after surgery was 4.5 years (range 0.2-5.6). Three-year RFS in intention to treat population was 72.3% (90% confidence interval [CI] 59.9-81.5), suggesting that the primary endpoint was met, and 3-year overall survival was 85.0% (90% CI 73.9-91.6). Grade 3 or higher adverse event with an incidence 10% of greater were neutropenia (13.2%), anorexia (13.2%), and diarrhea (10.5%). There was no treatment-related death. Conclusions: Adjuvant S-1 showed promising efficacy with manageable safety profile for patients with resectable ESCC after NAC-S, and warrants further evaluation in larger studies. Clinical trial information: UMIN000020204.