Chemotherapy is an important treatment modality for gastric cancer (GC); however, it usually fails because of drug resistance, especially multidrug resistance (MDR). Previously, we found a novel subset of MDR-associated microRNAs (miRNAs) through high-throughput functional screening. In this report, we investigated the exact roles and mechanisms of miR-23b-3p in the MDR of GC. Using gain or loss-of-function in in vitro and in vivo experiments, we found that overexpression of miR-23b-3p reversed cancer cell resistance to multiple chemotherapeutics in vitro and sensitize tumors to chemotherapy in vivo. Reporter gene assay and western blot analysis showed that ATG12 and HMGB2 were the direct targets of miR-23b-3p. Meanwhile, ATG12 and HMGB2 were positively associated with the occurrence of autophagy. Reducing the expression of these target genes by siRNA or inhibition of autophagy both sensitized GC cells to chemotherapy. These findings suggest that a miR-23b-3p/ATG12/HMGB2/autophagy-regulatory loop has a critical role in MDR in GC. In addition, miR-23b-3p could be used as a prognostic factor for overall survival in GC. In conclusion, our data demonstrated that miR-23b-3p inhibited autophagy mediated by ATG12 and HMGB2 and sensitized GC cells to chemotherapy, and suggested the potential application of miR-23b-3p in drug resistance prediction and treatment.