It is known that miR-381 plays a therapeutic role in a variety of cancers, but the regulatory mechanism of miR-381 in the treatment of lung cancer remains unclear. This study is aimed at exploring the expression level and mechanism of miR-381 in lung cancer. In this experiment, quantitative real-time PCR (qRT-PCR), western blot, and other methods were used to detect the expression of miR-381 and ubiquitin-specific protease 39 (USP39) in lung cancer tissues. The target genes of miR-381 were predicted by bioinformatics techniques, and the targeting relationship between miR-381 and USP39 was verified by the dual-luciferase reporting method. The expression levels of miR-381 and USP39 were adjusted to verify the effect of miR-381 on the expression of USP39 gene. The effect of miR-381 expression on proliferation of lung cancer cells was verified by cell proliferation and invasion experiments. miR-381 was downregulated in non-small-cell lung cancer tissues and cell lines, while USP39 was upregulated. The dual-luciferase reporter gene assay showed that miR-381 and USP39 had targeted binding sites. After transfection with miR-381 mimics, USP39 expression was significantly decreased, cell proliferation decreased, and apoptosis increased. After transfection with miR-381 inhibitor, USP39 expression was significantly increased, cell proliferation increased, and cell apoptosis decreased. Overexpression of USP39 significantly increased the invasion ability and cell survival curve ( p < 0.05 ). In conclusion, overexpression of miR-381 can regulate the expression of USP39, inhibit the proliferation and invasion of cancer cells, and induce apoptosis of cancer cells. This may provide a new perspective and strategy for targeted therapy of non-small-cell lung cancer.