MicroRNAs have emerged as fundamental regulators in gene expression through silencing gene expression at the post-transcriptional and translational levels. In this study, miR-143 expression and biological functions in AGS/MNK28 cell lines was investigated. Results indicated that the expression of miR-143 was significantly down-regulated in cancer tissues and in gastric cancer (GC) cell lines. Target prediction algorithms (Target Scan and miRanda) showed that GABARAPL1 was a potential target gene of miR-143. GABARAPL1, also regarded as autophagy-related protein 8 (Atg8) is a ubiquitin-like protein required for the formation of autophagosomal membranes. Then, several different assays were conducted to detect autophagy in AGS/MNK28 after transfected with miR-143. In the present study, miR-143 was firstly identified as a autophagy inhibitor in GC cells via targeting GABARAPL1. Quercetin is one of the most prominent dietary antioxidants in human diet and lately it is grabbing some serious attention as a potentially powerful cancer fighter. However, the effect of Quercetin was unexpected decreased in GC cells on account of the appearance of Quercetin-induced autophagy. Therefore, applicable autophagy inhibitors might enhance the chemosensitivity of Quercetin. Furthermore, the therapeutic response of Quercetin in the combination of miR-143 was evaluated by MTT, Hochest and western blot, results suggesting that the chemosensitivity of Quercetin was enhanced when in combination with miR-143 in AGS/MNK28 cells. In conclusion, we determined miR-143 as a potent inhibitor of autophagy via targeting GABARAPL1 and miR-143 could improve the efficacy of Quercetin though autophagy inhibition in GC cell lines, thus representing a novel potential therapeutic target for gastric cancer.