Cancer stem cells (CSCs) have the ability to self-renew and differentiate to give rise to heterogeneous phenotype of the tumor cells. It is believed that these cells are involved in metastasis, recurrence and therapy resistance in various cancers. CSCs have been identified in prostate cancer (PCa), one of the most diagnosed malignancies in men over the world, for which chemotherapy resistance is a major problem in the treatment of castration-resistant advanced stages. Molecular signatures, gene expression and functional features have been reported for PCa CSCs. Most data come from cell lines which may not represent the actual tumor. In the present work, a CSCs enriched population obtained from PCa explants was functionally characterized and analyzed for drug resistance. Tumorsphere cultures positive for ABCG2 transporter, CD133, CD44, cytokeratins 5 and 18 (CK5 and CK18) and negatives for androgen receptor (AR) and prostate-specific antigen (PSA) showed higher clonogenic capacity, holoclone-forming ability, colony-forming capacity in soft agar and lower proliferative and apoptotic rate than control adherent cell cultures. Furthermore, exposing tumorsphere cultures to ABCG2 substrate drugs resulted in a high survival rate compared with control PCa cells. This high drug resistance was decreased using a selective inhibitor of ABCG2. According to these results, tumorspheres from PCa explants showed a functional stem phenotype and a marked drug resistance, probably mediated by high expression of the ABCG2 transporter, which might be considered as a suitable therapeutic target for CSCs.