We previously reported that p21, a cell cycle regulator, plays important roles in chronic hypertensive renal injury. However, the roles of renal p21 in acute kidney injury, a life-threatening disease that can occur independently of the pathological background of patients (whether renal p21 is up-regulated or not), have not been fully clarified yet. In the present study, we evaluated the role of p21 in acute kidney injury and the effects of ischemic preconditioning (IPC). The mice lacking functional p21 (p21-KO) and its wild-type control underwent renal ischemia followed by reperfusion (I/R) with or without ischemic preconditioning. IPC attenuated I/R injury in wild-type mice, but not in p21-KO mice. Moreover, IPC increased the renal expression of p21 prior to I/R compared with sham surgery (p21/β-actin, sham: 1.00±0.18 fold, IPC: 4.12±0.32 fold, n=6, p<0.01). Immunohistochemisty (IHC) for p21 showed that there was an increase in the number of p21-positive tubular cells in mice that underwent I/R with and without IPC, and that the p21 immunoreactivity was increased in the nuclei of tubular cells, suggesting the effects on cell cycle. IPC decreased the number of proliferating tubular cells before I/R (0.24±0.11 fold vs. sham group, p<0.01) and increased it at 24 h after I/R (1.66±0.03 fold vs. sham group, p<0.01) in the kidney of wild-type mice. In p21-KO mice, IPC did not change the number of proliferating cells before I/R (0.99±0.10 fold, vs. sham group), and decreased it after I/R (0.53±0.08 fold, vs. sham group, p<0.01). Flow cytometry showed that IPC increased the number of cells in the G1 phase of cell cycle before I/R compared with sham surgery. Additionally, intravital imaging in combination with the mice that express mKO2, a fluorescent protein, at G1 phase of cell cycle showed that IPC increased the number of mKO2-positive cells in the proximal tubules. In conclusion, renal p21 is essential for the beneficial effects of renal IPC. Transient cell cycle arrest by IPC through a p21-dependent pathway seems to be important for recovering tubular cell proliferation after I/R.