Diabetes mellitus is a major health problem that affects a patient’s life quality throughout the world due to its worst complications. It was recognized that chronic hyperglycemia with oxidative stress was the major cause of male infertility. Echinacea purpurea ethanol extract (EE) contains phenolic acid and isobutylamides had been proven to ameliorate diabetic complications. Chitosan/silica nanoparticles are well-known in the medicinal field because of its controlled release and drug delivery properties. This study was aimed at investigating whether the EE encapsulated chitosan/silica nanoparticle (nano-EE) can enhance the amelioration of male infertility. Our results indicated that the average size of nano-EE was 218 ± 42 nm with an encapsulation efficiency of 66.9% and loading capacity of 39.9%. The reduction in oxidative stress and antioxidant activity of nano-EE was observed in LC-540 cells. In in vivo experiment, 33 mg/kg of streptozotocin (STZ) was used to induce diabetes in male Sprague-Dawley rats. Diabetic rats were treated with nano (465 mg/kg), nano-EE 1 (93mg/kg), nano-EE3 (279mg/kg), nano-EE5 (465 mg/kg), and metformin (Met) (200 mg/kg) for 7 weeks. The results show that the nano-EE5 can improve hyperglycemia, insulin resistance, and plasma fibroblast growth factor 21 (FGF 21) resistance. It was also confirmed that nano-EE5 significantly improved the testis tissue structure, increasing sperm quality and DNA integrity as well as reducing reactive oxygen species level.