microRNA (miR)-381 is downregulated and exhibits anti-invasive activity in non-small cell lung cancer (NSCLC). In this study, we investigated the role of miR-381 in proliferation, tumorigenesis, and cisplatin resistance of NSCLC cells. The effects of miR-381 overexpression on proliferation, tumorigenesis, cell cycle progression, and cisplatin sensitivity were examined. Overexpression of miR-381 significantly inhibited cell proliferation and colony formation in vitro and tumorigenesis in vivo. Ectopic expression of miR-381 arrested NSCLC cells at G0/G1 phase, which was accompanied by increased expression of p21 and p27 and decreased expression of cyclin D1 and CDK4. Compared to A549 parental cells, cisplatin-resistant equivalents (A549/CDDP) had reduced levels of miR-381. miR-381 re-sensitized A549/CDDP cells to cisplatin and potentiated cisplatin-induced apoptosis. Mechanistically, miR-381 interfered with the activation of nuclear factor (NF)-κB through repression of inhibitor of differentiation 1 (ID1). Co-expression of ID1 reversed the suppression of proliferation and enhancement of cisplatin cytotoxicity by miR-381. Taken together, miR-381 can induce growth suppression and chemosensitization in NSCLC, largely through inactivation of NF-κB via downregulation of ID1. Restoration of miR-381 represents a potential therapeutic strategy for NSCLC.