Objective To explore the expression of USP39 in cancer tissues of non-small cell lung cancer (NSCLC) and its clinical significance both in vivo and in vitro. Methods A total of 85 patients with NSCLC were involved in this study. Expressions of USP39 were identified by Immunohistochemistry. Statistical methods were performed to determine the clinical relevance of USP39 in NSCLC. Quantitative real-time PCR was conducted to detect the expression of UPS39 in NSCLC cell lines and BEAS-2B cells. CCK-8 and Flow cytometry were carried out to measure cell proliferation and cell apoptosis. Results Positive rate of USP39 was upregulated in NSCLC tissues compared with adjacent normal tissue. Expression of USP39 was closely associated with TNM stage in NSCLC patients. USP39 was upregulated in NSCLC cell line than that in BEAS-2B cells. Cell proliferation was decreased while cell apoptosis rate was increased in NSCLC cells transfected with USP39 siRNAs when compared with those in USP39 NC group. Furthermore, cell cycle was arrested at G1 stage in NSCLC transfected with USP39 siRNAs Conclusions USP39 expression is upregulated in NSCLC and closely associated with disease prognosis.