AbstractPatients with castration-resistant prostate cancer (CRPC) often develop drug resistance after treatment with enzalutamide. The goal of our study was to identify the key genes related to enzalutamide resistance in CRPC and to provide new gene targets for future research on improving the efficacy of enzalutamide. Differential expression genes (DEGs) associated with enzalutamide were obtained from the GSE151083 and GSE150807 datasets. We used R software, the DAVID database, protein–protein interaction networks, the Cytoscape program, and Gene Set Cancer Analysis for data analysis. The effect ofRAD51knockdown on prostate cancer (PCa) cell lines was demonstrated using Cell Counting Kit-8, clone formation, and transwell migration experiments. Six hub genes with prognostic values were screened (RAD51,BLM,DTL,RFC2,APOE, andEXO1), which were significantly associated with immune cell infiltration in PCa. HighRAD51,BLM,EXO1, andRFC2expression was associated with androgen receptor signaling pathway activation. Except forAPOE, high expression of hub genes showed a significant negative correlation with the IC50 of Navitoclax and NPK76-II-72-1.RAD51knockdown inhibited the proliferation and migration of PC3 and DU145 cell lines and promoted apoptosis. Additionally, 22Rv1 cell proliferation was more significantly inhibited withRAD51knockdown than withoutRAD51knockdown under enzalutamide treatment. Overall, six key genes associated with enzalutamide resistance were screened (RAD51,BLM,DTL,RFC2,APOE,andEXO1), which are potential therapeutic targets for enzalutamide-resistant PCa in the future.