Gastric cancer (GC), among the most prevalent malignant tumors globally, demonstrates a rapid metastasis rate leading to high mortality. While microRNAs (miRNAs) have been recognized as critical regulators of tumor progression, the specific role of miR-28-3p in GC remains unclear. In this study, we demonstrate that miR-28-3p acts as a tumor suppressor by inhibiting GC cell proliferation and EMT-driven migration in vitro, as well as tumor growth and metastasis in vivo. Mechanistically, miR-28-3p directly targets ADP ribosylation factor 6 (ARF6), a small GTPase identified as an oncogene in GC. We reveal that ARF6 is significantly upregulated in GC and activates the GLI1/2-dependent Hedgehog signaling pathway, promoting tumor growth and EMT. Notably, ARF6 knockdown mitigates the pro-tumor effects caused by miR-28-3p deficiency, while combined ARF6 inhibition and Hedgehog pathway suppression exhibit synergistic anti-tumor effects. This study establishes the miR-28-3p-ARF6-Hedgehog signaling axis as a critical regulatory pathway in GC progression. Our findings provide novel insights into GC pathogenesis and highlight the therapeutic potential of targeting this axis for innovative treatment strategies.Copyright © 2025 The Authors. Published by Elsevier Ltd.. All rights reserved.