S-1 has been recommended as adjuvant chemotherapy in patients after curative surgery for gastric cancer. However, some patients suffer recurrence even after S-1 adjuvant chemotherapy. The present study was conducted to find a predictive marker of the efficacy of S-1 adjuvant chemotherapy. We examined the microRNA (miRNA) expression of 35 patients who underwent S-1 adjuvant chemotherapy after curative surgery (R0) for pathological stage II or III gastric cancer. miRNAs were extracted from formalin-fixed, paraffin-embedded specimens for analysis and miRNA expression was examined using miRNA oligo chips. Fifteen patients relapsed and 20 did not over 5 years. Five miRNAs (miR-92b, 422a, 4732-5p, 4758-3p and 221) were highly expressed according to the tumor/normal (T/N) ratio in the patients who relapsed but not in those who did not relapse (P-value <0.05) by microarray analysis. If tumors showed high expression of 4 miRNAs (miR-92b, 422a, 4732-5p and 4758-3p) their positive predictive value of relapse was 93.8% and negative predictive value was 92.3%. In this case, their disease-free survival rate and overall survival rate were very poor. Our findings indicate that miR-92b, miR‑422a, miR-4732-5p and miR-4758-3p are closely associated with relapse following S-1 adjuvant chemotherapy in gastric cancer.