Purpose: The organic anion transporter 0ATP1B3, encoded by SLC01B3, is involved in the transport of steroid hormones. However, its role in testosterone uptake and clinical outcome of prostatic cancer is unknown. This study examined (a) the SLC01B3 genotype in cancer cells as well as the uptake of testosterone by cells transfected with genetic variants of SLC01B3; (b) the expression of 0ATP163 in normal prostate, benign prostatic hyperplasia, and prostatic cancer; and (c) the role of SLC01B3 haplotype on clinical outcome of Caucasian patients with androgen-independent prostatic cancer. Experimental Design: SLC01B3 genotype was assessed in the NCI-60 panel of tumor cells by sequencing, whereas testosterone transport was analyzed in Cos-7 cells transfected with WT, 334G, and 699A SLC01B3 variants. 0ATP1B3 expression in prostatic tissues was examined by fluorescence microscopy, and the relationship between SLC01B3 haplotypes and survival was examined in patients. Results: Cells transfected with wild-type (334T/699G) SLC01B3, or with a vector containing either the 334G or 699A variants, actively transported testosterone, whereas its uptake was impaired in cells transfected with a gene carrying both 334G and 699A single nucleotide polymorphisms. Prostatic cancer overexpresses 0ATP1B3 compared with normal or benign hyperplastic tissue; patients with SLC01B3 334GG/699AA haplotype showed longer median survival (8.5 versus 6.4 years; P = 0.020) and improved survival probability at 10 years (42% versus 23%; P < 0.023) than patients carrying TT/AA and TG/GA haplotypes. Conclusions: The common SLC01B3 GG/AA haplotype is associated with impaired testosterone transport and improved survival in patients with prostatic cancer.