Aldehyde dehydrogenase 1 (ALDH1) characterizes tumor-initiating cells in solid tumors; however, little is known about its expression in intratumoral stromal cells. Herein, we aimed to dissect its potential dual relevance in prostate cancer (PCa). ALDH1 expression was evaluated immunohistochemically in tumor and stromal cells in primary PCa and metastases. It was correlated to clinico-pathologic parameters, patients' outcome, and selected proteins (CK5/6, CK14, CK8/18, CK19, EpCAM, Ki-67, E-cadherin, N-cadherin, and vimentin). ALDH1 protein was detected in tumor and stromal cells in 16% and 67% of 348 primary PCa, respectively. Tumor cell ALDH1 expression was associated with advanced T stage (P = 0.009), higher Gleason score (P = 0.016), shorter time to biochemical recurrence (TBR P = 0.010) and CK14 expression (P = 0.023). Stromal cell ALDH1 expression correlated to lower T stage (P = 0.008) and Gleason score (P = 0.016), N0 stage (P = 0.017), and longer TBR (P = 0.017). It occurred to be an independent predictor of good prognosis in the subgroup of d'Amico high-risk patients (multivariate analysis, P = 0.050). ALDH1-positive stromal cells were found in tumors characterized frequently by CK8/18 (P = 0.033) or EpCAM expression (P < 0.001) and rarely by epithelial-mesenchymal transition defined as CK8/18(-)vimentin(+) phenotype (P = 0.003). ALDH1-positive tumor and stromal cells were detected in 33% and 41% of hormone naive lymph node metastases (n = 63), 52% and 24% of castration resistant bone metastases, as well as 89% and 28% of castration resistant visceral metastases (n = 21), respectively. We have determined that contrary to tumor cell ALDH1, the presence of stromal ALDH1 is associated with epithelial phenotype of primary PCa, improved clinical outcome, and is less frequent in PCa metastases.