As one of the most frequent diabetic complications, diabetic foot ulcer (DFU) can cause limb ischemia or even amputation. Paeoniflorin (PF) has been reported to possess many kinds of biological functions, such as antioxidant and anti-inflammatory effects. However, the role of PF in DFU remains unknown. In this study, streptozotocin (STZ)-induced diabetic rat models and high glucose (HG)-treated Human immortalized keratinocytes (HaCaT) cells were established. Histological analysis, immunohistochemistry, Electrophoretic mobility shift assay, MTT assay, TUNEL assay, oxidative stress analysis, ELISA assay and western blot were used to investigate the role and underlying mechanisms of PF on healing in DFU. Our results showed that the STZ-induced diabetic rats had delayed wound healing compared with the normal rats, exhibited by intense oxidative DNA damage, low vascular endothelial growth factor (VEGF) and transforming growth factor β1 (TGF-β1) expression, as well as increased apoptosis. PF treatment activated the expression of nuclear factor-E2-related factor 2 (Nrf2) and improved wound healing in DFU rats. Our in vitro experiments confirmed that PF accelerated wound healing through the Nrf2 pathway under hyperglycemic conditions, with alleviated oxidative stress, increased cell proliferation and migration, decreased apoptosis, and increased the expression of VEGF and TGF-β1. Our study demonstrates the therapeutic benefits of PF in diabetic wound healing, which provides a reference for future clinical trials using PF in DFU treatment.