The hypothalamic kisspeptin signaling system is a major positive regulator of the reproductive neuroendocrine axis, and loss of Kiss1 in the mouse results in infertility; a condition generally attributed to its hypogonadotropic hypogonadism. We demonstrate that in Kiss1-/- female mice, acute replacement of gonadotropins and estradiol restores ovulation, mating and fertilization; however, these mice are still unable to achieve pregnancy because embryos fail to implant. Progesterone treatment did not overcome this defect. Kiss1+/- embryos transferred to a wild-type female mouse can successfully implant, demonstrating the defect is due to maternal factors. Kisspeptin and its receptor are expressed in the mouse uterus, and we suggest that it is the absence of uterine kisspeptin signaling that underlies implantation failure. This absence, however, does not prevent the closure of the uterine implantation chamber, proper alignment of the embryo and the ability of the uterus to undergo decidualization. Instead, the loss of Kiss1 expression specifically disrupts embryo attachment to the uterus. We observed that on the day of implantation, Lif, a cytokine that is absolutely required for implantation in mice, is weakly expressed in Kiss1-/- uterine glands and that the administration of exogenous Lif to hormone-primed Kiss1-/- female mice is sufficient to partially rescue implantation. Taken together, our study reveals that uterine kisspeptin signaling regulates glandular Lif expression, thereby identifying a novel and critical role for kisspeptin in regulating embryo implantation in the mouse. This study provides compelling reasons to explore this role in other species, particularly livestock and humans.