To gain an understanding of the mechanisms by which Leydig cell steroidogenic function degenerates with ageing, we explored steroidogenic gene expression in relation to antioxidation status and endoplasmic reticulum (ER) stress during the ageing of mice. Expression of StAR, P450scc and other steroidogenic enzymes decreased starting at middle age (12-month-old) compared to that of the young control (3-month-old) mice. The immunohistochemical staining intensity of 3β-HSD for Leydig cells was significantly weaker in the aged (24-month-old) group than that in the young control group. The number of Leydig cells showed no significant difference between the groups. A progressive reduction in antioxidants MnSOD and GPx4 was observed in the testicular tissue with down-regulated SIRT1 protein level in the middle-aged and aged (24-month-old) mice. The number of testicular macrophages was significantly higher in the aged group than that in the middle-aged and young mice. Age-associated up-regulation of ER stress markers such as GRP78 and Chop was observed. These results suggested that oxidative stress and ER stress might play a role in the deficit of Leydig cell steroidogenic function during ageing.