Background: The cell cycle gene centromere protein K (CENPK) is upregulated in various cancers; however, the clinical value and mechanism of CENPK in prostate cancer (PCa) and castration-resistant prostate cancer (CRPC) remain unclear. Methods: The expression of CENPK in PCa was analyzed in both patients with PCa and cell lines using immunohistochemistry (IHC), real-time quantitative reverse transcription PCR (qRT-PCR), Western blot and bioinformatics analyses. Knockdown of CENPK in PCa cells was achieved by transfecting siRNAs and assessed using qRT-PCR and Western blotting. MTT and colony formation assays were used to assess the growth of PCa cells. The cell cycle was analyzed using propidium iodide (PI) staining and flow cytometry. To study the possible biological function of CENPK, pathway enrichment analysis was performed by dividing these groups into a high CENPK expression group and a low CENPK expression group based on the median CENPK expression level. Finally, the correlation between CENPK expression in PCa and clinical parameters was evaluated. Results: Our study revealed that CENPK was expressed at high levels in CRPC tissues and cell lines compared to primary PCa. The downregulation of CENPK significantly inhibited cell viability and reduced the number of colonies formed by LNCaP-AI and DU145 cells (two CRPC cell lines). Gene enrichment and flow cytometry analyses showed that high CENPK expression was linked to mitotic spindles and the cell cycle and may be involved in mitosis in the cell cycle of cancer cells to modulate cell proliferation and promote the development of CRPC. Moreover, patients exhibiting higher expression of the CENPK mRNA experienced shorter disease-free survival (DFS) and overall survival (OS) than the lower expression group. Conclusions: This study provides novel molecular insights into the role of CENPK in castration-resistant PCa cells and reveals that an increase in CENPK expression may indicate shorter DFS and a poor prognosis for patients with PCa. Targeting CENPK may be a novel strategy for the treatment of PCa.