Aldehyde dehydrogenase 1A3 (ALDH1A3) has been implicated in the survival and proliferation of prostate cancer cells. We retrospectively reviewed our patients with advanced disease on adjuvant hormonal therapy after prostatectomy. Time to castration resistance stage was documented. And Immunohistochemistry analysis for ALDH1A3 was performed for those patient samples on tissue microarray. Using bioinformatics analysis for RNA sequencing data of both primary prostate cancer and metastatic castration resistance prostate cancer (mCRPC) from online datasets, we have found that the expression level of ALDH1A3 is lower in mCRPC group than in primary cancer group. Crispr-Cas9 was used to knock out ALDH1A3 in prostate cancer luminal cells, and morphologic analysis as well as the Gene Set Enrichment Analysis (GSEA) were facilitated to discover the mechanisms of the resistance phenotype. We found that the patients with ALDH1A3 low expression had shorter time to progression to castration resistance compared with those of higher expression group on adjuvant hormonal therapy after radical prostatectomy. The ALDH1A3 knockout cells gradually acquired resistance to androgen deprivation therapy, few cells have been found in knockout group showing as that the spindle-like luminal cells in charcoal stripped medium. Furthermore, PI3K pathway activation has been confirmed by Western blot. The PI3K pathway inhibitor BEZ235 has been demonstrated that the acquired ADT resistance by ALDH1A3 down regulation could be rescued by PI3K pathway inhibitor. Together, these results suggested a novel function for ALDH1A3 in development of mCRPC, and indicated PI3K pathway inhibitor has the potential in the treatment of a subgroup of mCRPC patients.