The high disability, mortality and morbidity of diabetic ulcers make it urgent to explore effective strategies for diabetic wound repair. TrxR1 plays a vital role in regulating redox homeostasis in various pathologies. In the present study, the effect of berberine (BBR) on diabetic wounds was investigated in streptozotocin (STZ)-induced diabetic rats and a high glucose (HG)-induced cell model, and the mechanism of BBR on TrxR1 was elucidated. BBR treatment remarkably accelerated wound healing and enhanced extracellular matrix (ECM) synthesis and significantly inhibited HG-induced HaCaT cell damage. Further analysis indicated that BBR activated TrxR1, suppressed its downstream JNK signaling, thereby inhibiting oxidative stress and apoptosis, promoted cell proliferation, down-regulated matrix metalloproteinase (MMP) 9 (MMP9) and up-regulated transforming growth factor-β1 (TGF-β1) and tissue inhibitors of MMP 1 (TIMP1), resulting in accelerated wound healing. Importantly, the enhancement of BBR on wound repair was further abolished by TrxR1 inhibitor. Moreover, in diabetic wounds induced by a combination of STZ injection and high-fat diet, BBR significantly increased wound closure rate and TrxR1 expression, and this was reversed by TrxR1 inhibitor. These data indicated that topical BBR treatment accelerated diabetic wound healing by activating TrxR1. Targeting TrxR1 may be a novel, effective strategy for restoring redox homeostasis and promoting diabetic wound healing.