The Na⁺-dependent taurocholate co-transporting polypeptide (NTCP/SLC10A1) is a hepatocyte specific solute carrier, which plays an important role in maintaining bile salt homeostasis in mammals. The absence of an hepatic Na⁺-dependent bile salt transport system in marine skate and rainbow trout raises a question regarding the function of the Slc10a1 gene in these species. Here we have characterized the Slc10a1 gene in the marine skate, Leucoraja erinacea. The transcript of skate Slc10a1 (skSlc10a1) encodes 319 amino acids and shares 46% identity to human NTCP (hNTCP) with similar topology to mammalian NTCP. SkSlc10a1 mRNA was mostly confined to the brain and testes with minimal expression in the liver. An FXR-bile salt reporter assay indicated that skSlc10a1 transported taurocholic acid (TCA) and scymnol sulfate, but not as effectively as hNTCP. A ³H-TCA uptake assay revealed that skSlc10a1 functioned as a Na⁺-dependent transporter but with low affinity for TCA (K_m=92.4 µM) and scymnol sulfate (K_i=31 µM), compared to hNTCP (TCA, K_m=5.4 µM; Scymnol sulfate, K_i=3.5 µM). In contrast, the bile salt concentration in skate plasma is 2 µM, similar to levels seen in mammals. Interestingly, skSlc10a1 demonstrated transport activity for the neurosteroids DHEAS and estrone-3-sulfate at physiological concentration, similar to hNTCP. Together, our findings indicate that skSlc10a1 is not a physiological bile salt transporter, providing a molecular explanation for the absence of a hepatic Na⁺-dependent bile salt uptake system in skate. We speculate that Slc10a1 is a neurosteroid transporter in skate that gained its substrate specificity for bile salts later in vertebrate evolution.