Sodium taurocholate cotransporter polypeptide (NTCP), mainly expressed on the sinusoidal membrane of hepatocytes, is one of the major transporters responsible for liver bile acid (BA) re‐uptake. NTCP transports conjugated BA from the blood into hepatocytes and is crucial for correct enterohepatic circulation. Studies have shown that insufficient hepatic clearance of BA correlates with elevated serum BA in infants younger than 1 year of age. In the current study, we investigated human NTCP messenger RNA and protein expression by using reverse‐transcription quantitative polymerase chain reaction and immunoblotting in isolated and cryopreserved human hepatocytes from two different age groups, below and above 1 year of age. Here, we show that NTCP messenger RNA expression is not modulated whereas NTCP protein posttranslational glycosylation is modulated in an age‐dependent manner. These results were confirmed by quantification analysis of NTCP 55‐kDa N‐glycosylated bands, which showed significantly less total NTCP protein in donors below 1 year of age compared to donors older than 1 year. NTCP tissue localization was also analyzed by means of immunofluorescence. This revealed that NTCP cellular localization in fetal samples was mainly perinuclear, suggesting that NTCP is not glycosylated, while its postnatal localization on the plasma membrane is age dependent compared to multidrug resistant protein 2, which is apical starting in fetal life. Conclusion: After birth, the NTCP age‐dependent maturation process requires approximately 1 year to complete NTCP glycosylation in human hepatocytes. Therefore, NTCP late posttranslational glycosylation appears to be important for correct NTCP membrane localization, which might explain physiologic cholestasis in neonatal life and might play a central role for HBV infection after birth. (Hepatology Communications 2018;2:693‐702)