Nucleoside diphosphate kinase 4 (NME4) is abnormally expressed in a variety of cancer types. However, the function of the NME4 gene in non‑small cell lung cancer (NSCLC) remains to be elucidated. In order to investigate the role of NME4 in NSCLC, the present study detected the expression of the NME4 gene in the Cancer Genome Atlas database, and in BEAS‑2B, NCI‑H1299 and A549 cell lines. NME4 was significantly overexpressed in NSCLC tissues and NSCLC cell lines. Furthermore, lentivirus‑mediated knockdown vector infection, cell proliferation, cell cycle, apoptosis, colony formation and MTT assays were conducted to explore the effect of NME4 on NSCLC in vitro. After knockdown of NME4 with short hairpin RNA, the cell cycle was arrest at the G1 phase, and proliferation and colony formation were inhibited in the NCI‑H1299 and A549 cell lines. The present results suggested that NME4 may serve as a novel tumor promoter, capable of enhancing NSCLC progression by overcoming cell cycle arrest and promoting proliferation.