This study was designed to detect preputial separation and glans penis changes in normal growing SD rats. We examined the changes until 53 days after birth. The glans penis surface changed from a protruding os penis structure to a W shape, and from a W shape to a flattened surface with age. The protruding os penis structure changed gradually to the W shape from postnatal day (PND) 30, and all rats had the W shape by PND 35. The flattened surface was observed from PND 39, and all rats had this structure by PND 44. In all rats, the day of complete preputial separation exactly corresponded to the day of appearance of the flattened surface.