Renal ischemia-reperfusion injury (IRI) is a leading cause of acute kidney injury (AKI) resulting in an abrupt deterioration of kidney function. CXC chemokine ligand 16 (CXCL16) contributes significantly to the pathogenesis of renal injury. However, the signaling pathway mechanisms of CXCL16 in IRI-induced AKI remains obscured. In this study, we examined the role of the CXCL16/ Rho Associated Coiled-Coil Containing Protein Kinase-1 (ROCK1) signaling pathway in AKI induced by IRI. In vivo, CXCL16 was induced markedly after IRI. Mice treated with anti-CXCL16 antibody displayed less severe renal dysfunction and tubular injury in response to IRI compared with vehicle-treated mice. Inhibition of CXCL16 substantially reduced apoptotic cells and suppresses caspase-3 activation in the kidneys of mice following IRI. Additionally, CXCL16 inhibition profoundly decreased the production of TNF-α, IL-1β and IL-6 in the kidneys of mice post IRI. Furthermore, the level of ROCK1 protein was upregulated in the kidney in response to IRI, an effect that was abolished by CXCL16 inhibitor. Finally, treatment with Y-27632 (a ROCK1 inhibitor) attenuated deterioration of renal function and tubular damage of mice after IRI. Administration of Y-27632 ameliorated apoptosis in the IRI-treated kidneys of mice. In injured HK-2 cells, CXCL16 activated ROCK1 resulting in the upregulation of caspase-3 protein and pro-inflammatory molecules, which was abolished by Y-27632. In summary, our findings demonstrate that CXCL16/ROCK1 signaling pathway may play an important role in the pathogenesis of IRI-induced AKI.