Objectives: miR-375 is one of the highly expressed microRNAs (miRNAs) found in pancreatic islets of both humans and mice. In this study, we investigated functions of miRNA miR-375 in porcine pancreatic stem cells (PSC). Materials and methods: We transfected mimic and inhibitor of miR-375 in PSCs to measure functional roles of the microRNA and its effects on cell cycle proliferation and cell differentiation were determined. Luciferase assays were also performed to reveal the target gene of miR-375. Results: Overexpression of miR-375 suppressed proliferation, promoted apoptosis and inhibited differentiation into islet-like cells. PDK1 was identified as being a target of miR-375. Furthermore, we found that overexpression of miR-375 inhibited activation of the PDK1-AKT signalling pathway. Conclusion: miR-375 directly targeted PDK1 in porcine PSCs, suppressing cell proliferation and differentiation into islet-like cells.