Severe acute liver injury due to accidental or intentional acetaminophen overdose presents a major clinical dilemma often requiring liver transplantation. In the present study, liver regeneration after profound liver injury in mice challenged with acetaminophen was facilitated by the exogenous addition of ELR‐containing CXC chemokines such as macrophage inflammatory protein‐2 (MIP‐2), epithelial neutrophil‐activating protein‐78 (ENA‐78), or interleukin 8. Intravenous administration of ELR‐CXC chemokines or N‐acetyl‐cysteine (NAC) immediately after acetaminophen challenge in mice significantly reduced histological and biochemical markers of hepatic injury. However, when the intervention was delayed until 10 h after acetaminophen challenge, only ELR‐CXC chemokines significantly reduced liver injury and mouse mortality. The delayed addition of ELR‐CXC chemokines to cultured hepatocytes maintained the proliferation of these cells in a CXCR2‐dependent fashion after acetaminophen challenge whereas delayed NAC treatment did not. These observations demonstrate that ELR‐CXC chemokines represent novel hepatic regenerative factors that exhibit prolonged therapeutic effects after acetaminophen‐induced hepatotoxicity.—Hogaboam, C. M., Bone‐Larson, C. L., Steinhauser, M. L., Lukacs, N. W., Colletti, L. M., Simpson, K. J., Strieter, R. M., Kunkel, S. L. Novel CXCR2‐dependent liver regenerative qualities of ELR‐containing CXC chemokines. FASEB J. 13, 1565–1574 (1999)