The cyclin-dependent kinase inhibitor p21 plays important roles in chronic renal disorders; however, its roles in response to acute renal stress are unclear. Here we evaluated p21 in acute kidney injury and ischemic preconditioning using wild-type and p21 knockout mice that underwent renal ischemia followed by reperfusion. The decline in renal function and histological changes were worse in the knockout than in wild-type mice. Ischemia/reperfusion increased p21 expression in the kidney of wild-type mice compared with sham surgery, suggesting p21 may confer tolerance to ischemia/reperfusion injury. We next tested whether p21 is associated with the protective effect of ischemic preconditioning, an established method to reduce ischemia/reperfusion injury. Ischemic preconditioning attenuated ischemia/reperfusion injury in wild-type but not p21-knockout mice. This preconditioning decreased the number of proliferating tubular cells before but increased them at 24 h after ischemia/reperfusion in the kidneys of wild-type mice. In p21-knockout mice, ischemic preconditioning did not change the number of proliferating cells before but decreased them after ischemia/reperfusion. Ischemic preconditioning increased renal p21 expression and the number of cells in the G1 phase of the cell cycle before ischemia/reperfusion compared with sham surgery. Thus, renal p21 is essential for the beneficial effects of renal ischemic preconditioning. Transient cell cycle arrest induced by ischemic preconditioning by a p21-dependent pathway seems to be important for subsequent tubular cell proliferation after ischemia/reperfusion.