<b>INTRODUCTION</b> NSCLC is the leading cause for cancer-related deaths worldwide. New therapeutic targets are needed, as development of resistance to current treatment, such as platinum-based chemotherapy, is inevitable. The purpose of this study was to determine the functional relevance and therapeutic potential of cell division cycle associated 3 protein (CDCA3) in NSCLC. <b>METHODS</b> The expression of CDCA3 in squamous and nonsquamous NSCLC was investigated by using bioinformatics, Western blot analysis of matched tumor and normal tissue, and immunohistochemistry of a tissue microarray. The function of CDCA3 in NSCLC was determined by using several in vitro assays with small interfering RNA depleting CDCA3 in a panel of three immortalized human bronchial epithelial cell (HBEC) lines and seven NSCLC cell lines. <b>RESULTS</b> In this study, cell division cycle associated 3 gene (CDCA3) transcripts were identified as highly increased in NSCLC versus in nonmalignant tissue, with high levels of CDCA3 being associated with poor patient prognosis. CDCA3 protein was also increased in NSCLC tissue and expression was limited to tumor cells. CDCA3 expression was similarly increased in a panel of NSCLC cell lines compared with in three HBEC lines. Although depletion of CDCA3 in the HBEC lines did not affect cellular proliferation, depletion of CDCA3 expression markedly reduced the proliferation of all NSCLC cell lines. CDCA3 depletion caused a defective G2/M-phase cell cycle progression, upregulation of p21 independent of p53, and induction of cellular senescence. <b>CONCLUSIONS</b> Our findings highlight CDCA3 as a prognostic factor and potential novel therapeutic target in NSCLC through inhibition of tumor growth and promotion of tumor senescence.