A major atheroprotective functionality of high‐density lipoproteins (HDLs) is to promote “reverse cholesterol transport” (RCT). In this process, HDLs mediate the efflux and transport of cholesterol from peripheral cells and its subsequent transport to the liver for further metabolism and biliary excretion. We have previously demonstrated in cultured hepatocytes that P2Y₁₃ (purinergic receptor P2Y, G protein–coupled, 13) activation is essential for HDL uptake but the potential of P2Y₁₃ as a target to promote RCT has not been documented. Here, we show that P2Y₁₃‐deficient mice exhibited a decrease in hepatic HDL cholesterol uptake, hepatic cholesterol content, and biliary cholesterol output, although their plasma HDL and other lipid levels were normal. These changes translated into a substantial decrease in the rate of macrophage‐to‐feces RCT. Therefore, hallmark features of RCT are impaired in P2Y₁₃‐deficient mice. Furthermore, cangrelor, a partial agonist of P2Y₁₃, stimulated hepatic HDL uptake and biliary lipid secretions in normal mice and in mice with a targeted deletion of scavenger receptor class B type I (SR‐BI) in liver (hypomSR‐BI–knockout^liver) but had no effect in P2Y₁₃ knockout mice, which indicate that P2Y₁₃‐mediated HDL uptake pathway is independent of SR‐BI–mediated HDL selective cholesteryl ester uptake. Conclusion: These results establish P2Y₁₃ as an attractive novel target for modulating RCT and support the emerging view that steady‐state plasma HDL levels do not necessarily reflect the capacity of HDL to promote RCT. (HEPATOLOGY 2010)