Cell surface receptors for high-density lipoprotein (HDL) on hepatocytes are major partners in the regulation of cholesterol homeostasis. We have previously demonstrated on human hepatocytes that apolipoprotein A-I binding to an ectopic F₁-ATPase stimulates the production of extracellular ADP that activates a P2Y₁₃-mediated high-density lipoprotein (HDL) endocytosis pathway. However, P2Y₁₃-dependent signalling pathway has never been described yet. The current study demonstrates a major role of cytoskeleton reorganization in F₁-ATPase/P2Y₁₃-dependent HDL endocytosis under the control of the small GTPase RhoA and its effector ROCK I. Indeed human hepatocytes (HepG₂ cells) stimulated by ADP or AR-C69931MX (both P2Y₁₃ agonists) showed a high specific activation of RhoA; in addition, inhibition of Rho proteins by C3 exoenzyme impairs HDL endocytosis whereas a constitutively active form of RhoA stimulates HDL endocytosis at the same level as under F₁-ATPase/P2Y₁₃ activation. Pharmacological inhibition of ROCK activity decreased HDL endocytosis following stimulation by apoA-I (F₁-ATPase ligand), ADP or AR-C69931MX and specific siRNA ROCK I extinction prevented the stimulation of HDL endocytosis without effect of ROCK II extinction. The functional involvement of ROCK I downstream F₁-ATPase/P2Y₁₃ was confirmed by the strong enrichment of the membrane fraction in ROCK I and by the requirement of actin polymerization in hepatocyte HDL endocytosis. These results allow the identification of the molecular events downstream P2Y₁₃ receptor activation for a better understanding of hepatocyte HDL endocytosis, the latest step in reverse cholesterol transport.