Renal ischemia-reperfusion (I/R) injury ranks as the primary cause of acute renal injury with severe morbidity and mortality. Side population (SP) cells have recently drawn increasing attention due to their critical role in injury repair and regeneration. Unfortunately, the underlying mechanism involved in renal I/R remains poorly elucidated. Here, pronounced increases of stromal cell-derived factor-1 (SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) were substantiated in I/R kidneys from C57BL/6 mice subjected to clamp the bilateral renal pedicles to mimic renal ischemia. Similar up-regulation of them was also determined in SP cells upon simulated ischemia/reperfusion (SI/R). In contrast to non-SP cells, SP cells exhibited higher viability, apoptosis resistance, chemotaxis, and paracrine actions following SI/R treatment, and these were further enhanced after SDF-1 stimulation. Interestingly, blocking CXCR4 signaling with AMD3100 notably ameliorated the above effects. Mechanism analysis corroborated that SDF-1/CXCR4 further induced the expression of ATP-binding cassette transporter ABCG2, an essential element for SP-mediated kidney regeneration after renal I/R injury. Moreover, AMD3100 pretreatment strikingly attenuated ABCG2 elevation in SP cells. Additionally, sonic hedgehog (SHH)-Gli 1 signaling was involved in SDF-1/CXCR4-mediated ABCG2 expression. When SP cells pretreated with AMD3100 were intravenously injected into I/R mice, SP cell-mediated decreases in blood urea nitrogen, serum creatinine, and histological score of kidney were noticeably attenuated, indicating that blocking CXCR4 pathway mitigated the therapeutic function of SP cells in renal I/R injury. Together, this research suggests that SDF-1/CXCR4 axis might act, via Shh-Gli1-ABCG2 signaling, as a positive regulator of SP cell-based therapies for renal I/R by Shh-Gli 1-ABCG2 signaling.