Bone marrow-derived mesenchymal stem cells (BMSCs) transplantation can repair acute kidney injury (AKI), but with limited effect. We test the hypothesis that CXCR4 overexpression improves BMSCs repair ability and that this is related to increased release of cytokines. Hypoxia/re-oxygenation pretreated renal tubular epithelial cells (HR-RTECs) were used. BMSCs, null-BMSCs and CXCR4-BMSCs were co-cultured with HR-RTECs. The number of migrating BMSCs was counted. Proliferating cell nuclear antigen (PCNA) expression, cell death and expressions of cleaved Caspase-3 and Bcl-2 in the co-cultured HR-RTECs were measured. Cytokeratin 18 (CK18) expression and cytokine secretions for the BMSCs cultured with HR-RTEC supernatant were detected. BMSC homing, renal function, proliferation and cell death of tubular cells were assayed in the AKI mouse model. CXCR4-BMSCs showed a remarkable expression of CXCR4. Stromal cell-derived factor-1 (SDF-1) in the HR-RTEC supernatant was increased. Migration of BMSCs was CXCR4-dependent. Proportions of CK18⁺ cells in BMSCs, null-BMSCs and CXCR4-BMSCs showed no difference. However, CXCR4 overexpression in BMSCs stimulated secretion of BMP-7, HGF, and IL-10. The neutralizing anti-CXCR4 antibody AMD3100 abolished this. In the co-cultured HR-RTECs, proportion of PCNA⁺ cells and Bcl-2 expression were enhanced; however, proportion of Annexin V+ cells and expression of cleaved Caspase-3 were reduced. The in vivo study showed increased homing of CXCR4-BMSCs in kidneys, associated with improved renal function, reduced ATN scoring, accelerated mitogenic response of tubular cells, and reduced tubular cell death. The enhanced homing and paracrine actions of BMSCs with CXCR4 overexpression suggest beneficial effects of such cells in BMSC-based therapy of AKI.