Kisspeptin, encoded by Kiss1 gene, is considered to be a key regulator for controlling gonadotropin-releasing hormone (GnRH) secretion in rodents. Kiss1 knockout (KO) rats are infertile due to the absence of GnRH secretion in both sexes. It has been reported that GnRH promotes female sexual behavior (lordosis), but the relationship between kisspeptin and lordosis is not clear. The present study aims to clarify the role of kisspeptin in female sexual behaviors. Kiss1 KO females showed lordosis behavior and lordosis quotients were not significantly different from wild-type controls. Kiss1 KO males showed robust lordosis behavior, while the behavior is suppressed in wild-type males. It is plausible that kisspeptin plays a crucial role in defeminization of the brain controlling sexual behaviors during perinatal period. Kiss1 KO males showed a high plasma testosterone level at embryonic day 18 and postnatal day 0 similar to wild-type males, indicating that kisspeptin may not be involved in stimulating testosterone secretion during perinatal period. When Kiss1 KO rats were administered with 150 ng estradiol benzoate (EB) or 1 nmol kisspeptin at postnatal day 0, EB suppressed female sexual behavior during adulthood in male and female rats. Kisspeptin treatment suppressed female sexual behaviors in Kiss1 KO males not in females, suggesting that kisspeptin may have roles activated by perinatal testosterone secretion. The present study showed that kisspeptin is needed to suppress female sexual behaviors in male rats. Taken together, kisspeptin is most likely to involve in the process of brain defeminization at downstream of perinatal testosterone secretion in male rats.