Non-small cell lung cancer (NSCLC) is a leading cause of tumor mortality worldwide. Nevertheless, the molecular mechanisms revealing NSCLC progression are still unclear. 5'-Nucleotidase domain containing 2 (NT5DC2), as a member of the NT5DC family, contains a haloacid dehalogenase motif localized in the N-terminus of these proteins. NT5DC2 plays an essential role in cancer development. The purpose of the study was to explore NT5DC2's role in tumorigenesis and its potential mechanisms in NSCLC. Our findings showed that NT5DC2 expression was significantly up-regulated in clinical NSCLC tissues compared to the paired non-tumor tissues. Functionally, NT5DC2 knockdown in A549 and H1299 cells markedly reduced cell proliferation, migration and invasion. On the contrary, NT5DC2 over-expression promoted NSCLC cell proliferative, migrative and invasive capacities. Additionally, NT5DC2 down-regulation significantly induced the G2 cell cycle arrest and apoptosis in NSCLC cells. Mechanistically, p53 might be a target of NT5DC2. The expression of p53 was highly induced in NSCLC cells with NT5DC2 knockdown, and opposite result was detected when NT5DC2 was over-expressed. Importantly, we found that NT5DC2 knockdown-restrained cell proliferation and -induced apoptosis was almost abrogated by p53 down-regulation in NSCLC cells, demonstrating that NT5DC2-regulated cell proliferation and apoptotic cell death in NSCLC was p53-dependent. Finally, we confirmed that reducing NT5DC2 could inhibit NSCLC tumorigenesis and hepatic metastasis in vivo. Collectively, these results suggested that NT5DC2 may be a potential driver of NSCLC, providing a new therapeutic target for the clinical treatment of NSCLC.