Dysregulation of the cell cycle contributes to tumor progression. Cell division cycle‑associated 3 (CDCA3) is a known trigger of mitotic entry and has been demonstrated to be constitutively upregulated in tumors. It is therefore associated with carcinogenic properties reported in various cancers. However, the role of CDCA3 in prostate cancer is unclear. In the present study, western blotting and analysis of gene expression profiling datasets determined that CDCA3 expression was upregulated in prostate cancer and was associated with a poor prognosis. CDCA3 knockdown in DU145 and PC‑3 cells led to decreased cell proliferation and increased apoptosis, with increased protein expression levels of cleaved‑caspase3. Further experiments demonstrated that downregulated CDCA3 expression levels induced G0/G1 phase arrest, which was attributed to increased p21 protein expression levels and decreased cyclin D1 expression levels via the regulation of NF‑κB signaling proteins (NFκB‑p105/p50, IKKα/β, and pho‑NFκB‑p65). In conclusion, these results indicated that CDCA3 may serve a crucial role in prostate cancer and consequently, CDCA3 knockdown may be used as a potential therapeutic target.