Non-small cell lung cancer (NSCLC) has the highest morbidity and mortality worldwide. OTU deubiquitinase 5 (OTUD5), a deubiquitinating enzyme, can enhance the stability of p53 and programmed cell death 5 (PDCD5), a protein related to the apoptosis, by deubiquitination. This study aimed to explore the biological function and underlying mechanism of OTUD5 in NSCLC. Western blot and qRT-PCR were used to detect the expression of OTUD5 protein and mRNA in NSCLC tissues and cells, respectively. RNAi was adopted to construct an OTUD5 low-expression model while the plasmids overexpressing p53 and PDCD5 were used to establish the overexpression models, respectively. CCK-8 assay, transwell assay, and apoptosis assay were carried out to analyze the changes in the proliferation, migration, and chemoresistance of A549 and HCC827 cells. The mechanism of OTUD5 in NSCLC was studied by Western blot. Down-regulated OTUD5 in NSCLC tissues was significantly correlated to a poor prognosis. The knockdown of OTUD5 inactivated p53 and PDCD5, promoting the proliferation and metastasis of NSCLC cells while inhibiting their apoptosis. OTUD5 knockdown also enhanced the resistance of NSCLC cells to doxorubicin and cisplatin. OTUD5 acted as a tumor suppressor in NSCLC by regulating the p53 and PDCD5 pathways.