Human hepatic organic anion-transporting polypeptides (OATPs), including OATP1B1, 1B3, and 2B1, are expressed at the basolateral membrane of hepatocytes and mediate the uptake of a variety of compounds from blood into hepatocytes. The liver-specific OATPs are increasingly recognized as playing important roles in the pharmacokinetic (PK) of many drugs and thus, involved in the clinically significant drug-drug interactions (DDIs). However, the evaluation of the specific roles of individual OATPs in hepatocytes is challenging because of the lack of selective inhibitors and probe substrates for each OATP member. In the present study, the uptake activity of OATP1B3 was examined in human hepatocytes cultured up to 14 days using an in vitro uptake assay. The results showed that OATP-mediated uptake of rosuvastatin, a substrate for OATPs declined substantially in cultured human hepatocytes. In contrast, the uptake of OATP1B3-selective substrate telmisartan was not measureable at earlier culture periods, but became detectable on Day 7 and showed culture duration-dependent changes from Day 7 to 14. Quantitative polymerase chain reaction (qPCR) analyses illustrated that the OATP functional change was not correlated with messenger ribonucleic acid (mRNA) expression alteration in hepatocytes cultured for 3 hours or 7 days. The OATP1B3-mediated telmisartan uptake was also culture medium- and donor-dependent, and only observed in 3 of 5 lots of hepatocytes cultured in 2 of 3 media tested. These results show that using human hepatocytes cultured in certain conditions may provide an excellent addition to transfected cell lines as a way to distinguish OATP1B3 from other hepatic OATP family members, such as OATP1B1, to provide more understanding of OATP-mediated clinical DDI.