Studies have shown that miR-4317 is dysregulated in tumor, but the biologic role of miR-4317 in tumor development and progression remains unknown. The present study aimed to investigate the role of miR-4317 in human gastric cancer. Quantitative real-time PCR was used to quantify miR-4317 expression levels in clinical gastric cancer specimens and cell lines. MTT, colony formation and cell cycle assays were performed to identify the contributions of miR-4317 to cell proliferation in gastric cancer cell lines. The results showed that miR-4317 was significantly decreased in 17 clinical gastric cancer specimens compared with adjacent non-tumor stomach tissues. Forced expression of miR-4317 suppressed gastric cancer cell proliferation and blocked S-G2/M transition. Bioinformatics and dual-luciferase reporter assays confirmed that ZNF322 is a direct target of miR-4317. Silencing ZNF322 recapitulated the cellular and molecular effects seen upon miR-4317 overexpression. These findings indicate that miR-4317 represses the proliferation of gastric cancer cell, at least in part, by targeting and suppressing ZNF322 and that it may serve as a therapeutic target for gastric cancer treatment.