ABSTRACT Background: Sorafenib is an anticancer drug used in the treatment of unresectable hepatocellular carcinoma and advanced renal cell carcinoma. It is a substrate for the human OATP1B1. This study aimed to assess the role of OATP1B1 in transportation and uptake of sorafenib in hepatocellular carcinoma and how OATP1B1 affects the pharmacodynamics of sorafenib in vitro and in vivo. Methods: Sorafenib transport was measured in HepG2, HepG2-OATP1B1*1a, HepG2-OATP1B1*1b, HepG2-OATP1B1*15, LO2, LO2-OATP1B1*1a, LO2-OATP1B1*1b, and LO2-OATP1B1*15 cells, as well as in HepG2 cells transfected with miR-148a mimics. The cell viability and apoptosis rate of cells treated with sorafenib were evaluated. A liver cancer rat model was established to explore the pharmacokinetics and pharmacodynamics of sorafenib after overexpression of Oatp2. Results: Changes in expression and genetic mutations of OATP1B1 significantly affected the uptake of sorafenib in HepG2 and LO2 transgenic cells, and sorafenib uptake by HepG2 was higher than that by LO2. Genetic mutations of OATP1B1 significantly affected the cell viability and apoptosis rate of HepG2 cells after sorafenib treatment. Compared to control HepG2 cells, miR-148a mimic-transfected HepG2 cells had decreased sorafenib uptake. The inhibitory effect of sorafenib on cell growth was weakened. PCN significantly increased the expression of Oatp2 and affected the pharmacokinetics of sorafenib. Vascular endothelial growth factor levels and microvascular density in tumor-adjacent tissues decreased significantly, suggesting that increased Oatp2 expression improves the treatment effect of sorafenib in a rat model of liver cancer. Conclusions: OATP1B1 plays an important role in the pharmacokinetics and pharmacodynamics of sorafenib in hepatocellular carcinoma.