Nanotechnology has rapidly grown in various research fields to treat oxidative stress in diabetes, including medicine and phytomedicine. Previous research showed that Acalypha hispida has strong antioxidant activity in vitro and anti-hyperglycemic activity in vivo. However, the size reduction in the crude extract should be applied to decrease the doses and increase the efficacy. The research objective was to evaluate the nano-extract of A. hispida leaves on Cu,Zn-SOD antioxidant content in the pancreas of diabetic rats. The analysis of Cu,Zn-SOD antioxidant content was carried out using immunohistochemistry. Cu,Zn-SOD content of pancreas in diabetic rats (DMC) was significantly lower than in normal rats (NLC). The antioxidant content of Cu,Zn-SOD in MET, CAH, NAH3, and NAH6 groups was significantly higher than in the DMC group of the pancreas of diabetic rats. Nano extract of A. hispida showed a better effect in increasing Cu,Zn-SOD antioxidant content than crude extract. The result concluded that nano-extract of A. hispida leaves increased Cu,Zn-SOD antioxidant content in the pancreas of diabetic rats.