Hepatic stellate cells (HSC) and liver endothelial cells (LEC) migrate to sites of injury and perpetuate alcohol induced liver injury. High mobility group box 1 (HMGB1) is a protein released from the nucleus of injured cells that has been implicated as a proinflammatory mediator. We hypothesized that HMGB1 may be released from ethanol-stimulated liver parenchymal cells and contribute to HSC and LEC recruitment. Ethanol stimulation of rat hepatocytes and HepG2 cells resulted in translocation of HMGB1 from the nucleus as assessed by Western blot. HMGB1 protein levels were increased in the supernatant of ethanol-treated hepatocytes compared to vehicle-treated cells. Migration of both HSC and LEC was increased in response to conditioned media for ethanol-stimulated hepatocytes (CM_EtOH) compared to vehicle-stimulated hepatocytes (CM_VEH) (P<0.05). However, the effect of CM_EtOH on migration was almost entirely reversed by treatment with HMGB1-neutralizing antibody or when HepG2 cells were pre-transfected with HMGB1-siRNA compared to control siRNA transfected HepG2 cells (P<0.05). Recombinant HMGB1 (100 ng/mL) also stimulated migration of HSC and LEC compared to vehicle stimulation (P<0.05 for both HSC and LEC). HMGB1 stimulation of HSC increased the phosphorylation of Src and Erk and HMGB1-induced HSC migration was blocked by the Src inhibitor PP2, and Erk inhibitor, U0126. Hepatocytes release HMGB1 in response to ethanol with subsequent recruitment of HSC and LEC. This pathway has implications for HSC and LEC recruitment to sites of ethanol induced liver injury.