Objective ·To investigate the impact of KH-type splicing regulatory protein (KHSRP) on the proliferation of prostate cancer cells and the regulation of downstream gene expression, and explore the potential role and mechanism of KHSRP in the transition of prostate cancer from androgen-dependent to androgen-independent. Methods ·Recombinant lentivirus was used to infect androgen-dependent prostate cancer LNCaP cells and androgen-independent prostate cancer DU145 cells to establish stable cell lines with functional deficiency/acquisition of KHSRP, and the functional differences of KHSRP between the two cell types were compared. Western blotting was used to detect the expression levels of KHSRP, androgen receptor (AR), and ankyrin 3 (ANK3) in the stable cell lines. Cell proliferation assay, colony formation assay, and mouse xenograft assay were performed to assess the impact of KHSRP on the proliferation ability of LNCaP cells. RNA sequencing (RNA-seq) was performed to identify downstream genes affected by KHSRP, and the mRNA expression levels of these genes were measured by quantitative real-time PCR (RT-qPCR). The expression of ANK3 and KHSRP in prostate tissue samples and the difference of ANK3 expression in different prostate cancer cell lines were analyzed by combining Cancer Cell Line Encyclopedia (CCLE), The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Results ·GEO data analysis showed that KHSRP was upregulated in prostate cancer tissues compared to benign prostate tissues, suggesting its association with prostate tumorigenesis. Cell proliferation assay, colony formation assay, and mouse xenograft assay demonstrated a negative correlation between KHSRP expression and the proliferation ability of LNCaP cells, indicating that KHSRP can inhibit the proliferation of prostate cancer cells, with a stronger effect on LNCaP cells than on DU145 cells. Western blotting and RT-qPCR analysis of the stable LNCaP cell lines showed that KHSRP overexpression led to a decrease in AR protein level without affecting its mRNA level, suggesting that KHSRP can indirectly regulate AR protein level. RNA-seq and RT-qPCR results showed KHSRP was positively correlated with the expression of ANK3, a regulatory factor affecting AR protein stability, and subsequent Western blotting confirmed an increase in ANK3 protein expression after KHSRP overexpression. TCGA data analysis further supported the correlation between KHSRP and ANK3 mRNA expression. Interestingly, according to CCLE and GEO data, the expression of ANK3 was closely related to prostate cancer malignancy. Conclusion ·KHSRP may indirectly regulate AR protein stability through ANK3, thereby influencing the proliferation of androgen-dependent prostate cancer cells and mediating the altered responsiveness to androgen in prostate cancer cells.