The sodium-taurocholate (TC) cotransporting polypeptide Ntcp/NTCP mediates TC uptake across the sinusoidal membrane of hepatocytes. Previously, we demonstrated that nitric oxide (NO) inhibits TC uptake through S-nitrosylation of a cysteine residue. Our current aim was to determine which of the 8 cysteine residues of Ntcp is responsible for NO-mediated S-nitrosylation and inhibition of TC uptake. Thus, we tested the effect of NO on TC uptake in HuH7 cells transiently transfected with cysteine to alanine mutant Ntcp constructs. Of the eight mutants tested, only C44A Ntcp displayed decreased total and plasma membrane (PM) expression levels which were also reflected in decreased TC uptake. C266A Ntcp showed a decrease in TC uptake that was not explained by a decrease in PM expression or localization indicating that C266 is required for optimal uptake. We speculated that NO would target C266 since a previous report had shown the thiol reactive compound MTSET inhibits TC uptake by wild type NTCP but not by C266A NTCP. We confirmed that MTSET targets C266 of Ntcp but surprisingly, we found that C266 was not responsible for NO-mediated inhibition of TC uptake. Instead we found that C96 was targeted by NO since C96A Ntcp was insensitive to NO-mediated inhibition of TC uptake. We also found that WT but not C96A Ntcp is S-nitrosylated by NO suggesting that C96 is important in regulating Ntcp function in response to elevated levels of NO.