Kisspeptin, encoded by the Kiss1 gene, stimulates GnRH secretion and is therefore critical for sex steroid secretion at puberty and in adulthood. However, kisspeptin's role in regulating sex steroid secretion earlier in development is unexplored. In rodents, testosterone (T) levels are higher in prenatal and newborn males than females. We determined whether kisspeptin-Kiss1r and GnRH signaling plays a role in sexually dimorphic perinatal T secretion in mice. Our results demonstrate that 1) T levels in newborn males are elevated at 4 h but not 20 h after birth, but hypothalamic Kiss1 and neurokinin B (NKB) levels in males are not different between these time points (and both are lower than in females); 2) serum T levels in newborn Kiss1r knockout (KO) males are higher than in newborn females and similar to wild-type (WT) males; 3) perinatal hypothalamic progesterone receptor (Pgr) expression, which is dependent on circulating levels of gonadally produced T, is significantly higher in prenatal and newborn Kiss1r KO and WT males than similarly aged females; 4) multiple measures of testicular growth and function are not different between developing Kiss1r KO and WT mice until after postnatal d 5; and 5) GnRH neurons of newborn males do not exhibit high c-fos coexpression, and newborn hypogonadal (hpg) male mice (lacking GnRH) secrete elevated T, similar to newborn WT males. We conclude that, unlike in puberty and adulthood, elevated T secretion in prenatal and neonatal mice is independent of both kisspeptin and GnRH signaling, and the necessity of kisspeptin-Kiss1r signaling for testicular function is first apparent after d 5.