Peritoneal metastasis (PM) is the main cause of poor prognosis in patients with advanced gastric cancer (GC). Increasing evidence has suggested that cancer-associated EVs in body fluids may assist in the diagnosis and treatment of GC. Here, we investigated the role of GC-derived EVs in PM development. Our results demonstrate that expression of the tumor suppressor promyelocytic leukemia zinc finger (PLZF) is decreased in GC tissues and PM lesions from GC patients. PLZF suppression promoted migration and invasion of peritoneal mesothelial HMrSV5 cells, while PLZF overexpression suppressed HMrSV5 cell migration and invasion. Microarray analysis revealed significantly upregulated expression of several miRNAs in EVs isolated from GC patients with PM, including miR-544. The increased miR-544 expression was confirmed in GC tissues and PM-derived EVs. Transfection with miR-544 reduced PLZF expression in HMrSV5 cells, while miR-544 inhibition increased PLZF expression. Incubation of GC cells with peritoneal mesothelial HMrSV5 cells showed that miR-544 could be transferred from GC-derived EVs to peritoneal cells, where it suppressed the PLZF expression. These findings indicate that EV-mediated transfer of miR-544 decreases the PLZF expression in PM lesions, which suggests miR-544 could potentially serve as a diagnostic biomarker and therapeutic target for treatment of GC patients.