Gastric cancer (GC) was referred to a malignant tumor of the digestive tract originating from the epithelium of gastric mucosa. Transcription factor DLX5 was verified as an oncogene in various types of tumors, while miR-376a-3p was speculated as a tumor suppressor. Based on the bioinformatics database, we hypothesized that miR-376a participated in the regulation of GC development by targeting DLX5. Compared with adjacent tissue, a significant increase of DLX5 expression was determined in GC tissues, but the expression level is significantly reduced in miR-376a. Similar expression signature of DLX5 and miR-376a was also determined between 4 GC cells (HGC, SGC, MGC, and AGS cell lines) and GES cell line. The level of DLX5 was notably reduced in HGC and MGC cell lines after miR-376a-3p overexpression, and increased after miR-376a-3p inhibition. Then, the inhibition role of miR-376a-3p on DLX5 was further proved by dual-luciferase reporter assay. Gain-of-function experiments showed that upregulation of miR-376a-3p in GC cells could inhibit the ability of epithelial-mesenchymal transition, proliferation, and invasion, and enhance the GC cell apoptosis level. However, these roles of miR-376a-3p could be abolished by DLX5 overexpression. This study confirmed that reduction of miR-376a-3p expression level in GC cells would lead to the increase in cell growth and invasion, indicating that upregulation of miR-376a-3p might have a potential therapeutic role on GC.