The study is to investigate the effects of paeoniflorin (PA) on renal function in cyclophosphamide-induced mice. Mice were injected with intraperitoneal cyclophosphamide (CYP, 200 mg/kg) or saline respectively. Mice were treated with PA (15, 30 mg/kg/day) or vehicle for the next 7 days. Then, mice were sacrificed to analyze the biochemical, histological parameters and mechanism research. Our results shown that PA significantly decreased the urine levels of uric acid and creatinine, serum and kidney levels of cytokines such as interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), PA also obviously attenuated the histological changes of the kidney tissues caused by CYP. Moreover, Western blot demonstrated that PA increased the AMPK levels and inhibited NF-κB signaling pathway and apoptosis in CYP-stimulated kidney tissues. In conclusion, PA might be considered as an effective agent in the amelioration of the kidney toxicity resulting from CYP treatment.