Downstream of kinase 3 (DOK3) is involved primarily with immune cell infiltration. Recent research reported the role of DOK3 in tumor progression, with opposite effects in lung cancer and gliomas; however, its role in prostate cancer (PCa) remains elusive. This study aimed to explore the role of DOK3 in PCa and to determine the mechanisms involved.To investigate the functions and mechanisms of DOK3 in PCa, we performed bioinformatic and biofunctional analyses. Samples from patients with PCa were collected from West China Hospital, and 46 were selected for the final correlation analysis. A lentivirus-based short hairpin ribonucleic acid (shRNA) carrier was established for silencing DOK3. A series of experiments involving the cell counting kit-8, bromodeoxyuridine, and flow cytometry assays were performed to identify cell proliferation and apoptosis. Changes in biomarkers from the nuclear factor kappa B (NF-κB) signaling pathway were detected to verify the relationship between DOK3 and the NF-κB pathway. A subcutaneous xenograft mouse model was performed to examine phenotypes after knocking down DOK3 in vivo. Rescue experiments with DOK3 knockdown and NF-κB pathway activation were designed to verify regulating effects.DOK3 was up-regulated in PCa cell lines and tissues. In addition, a high level of DOK3 was predictive of higher pathological stages and worse prognoses. Similar results were observed with PCa patient samples. After silencing DOK3 in PCa cell lines 22RV1 and PC3, cell proliferation was significantly inhibited while apoptosis was promoted. Gene set enrichment analysis revealed that DOK3 function was enriched in the NF-κB pathway. Mechanism experiments determined that knockdown of DOK3 suppressed activation of the NF-κB pathway, increased the expressions of B-cell lymphoma-2 like 11 (BIM) and B-cell lymphoma-2 associated X (BAX), and decreased the expression of phosphorylated-P65 and X-linked inhibitor of apoptosis (XIAP). In the rescue experiments, pharmacological activation of NF-κB by tumor necrosis factor-α (TNF-α) partially recovered cell proliferation after the knockdown of DOK3.Our findings suggest that overexpression of DOK3 promotes PCa progression by activating the NF-κB signaling pathway.Copyright © 2023 The Chinese Medical Association, produced by Wolters Kluwer, Inc. under the CC-BY-NC-ND license.