The main function of Leydig cells in the testis is to synthesize steroid sex hormone testosterone.Because autophagy is very active in the Leydig cells, it has been proposed to be a nice model to study the physiological function of autophagy for a long time.However, the function and underlying mechanism of autophay in Leydig cells are still unknown.Here we show that autophagy is required for testosterone synthesis in Leydig cells.We specifically ablated autophagy related gene Atg5 or Atg7 in the Leydig cells by mating Atg5flox/flox and Atg7flox/flox mice with steroidogenic factor-1 (SF1)-Cre mice, and found that the testosterone levels in their serum dropped to the female level.The decreasing of the testosterone level was due to lack of lipid and cholesterol in the Leydig cells.Further experiments showed that proteins related with TG and cholesterol biogenesis in Atg7-knockout Leydig cells did not decreased,while the cholesterol absorption rate of Atg7-knockout Leydig cells dropped markedly.These results suggest that autophagy may participate in testosterone synthesis by mediating cholesterol uptake in Leydig cells.