Lung surfactant is secreted via exocytosis of lamellar bodies from alveolar epithelial type II cells. Whether micro-RNAs regulate lung surfactant secretion is unknown. Micro-RNA-375 (miR-375) has been shown to be involved in insulin secretion. In this article, we report that the overexpression of miR-375 inhibited lung surfactant secretion. However, miR-125a, miR-30a, miR-1, miR-382 and miR-101 did not influence lung surfactant secretion. miR-375 had no effects on surfactant synthesis or the formation of lamellar bodies. However, miR-375 did abolish the lung surfactant secretagogue-induced disassembly and reassembly of cytoskeleton. Our results suggest that miR-375 regulates surfactant secretion via the reorganization of cytoskeleton.