While hypothalamic kisspeptin (KP) neurons play well-established roles in the estrogen-dependent regulation of reproduction, little is known about extrahypothalamic KP-producing (KPLS) neurons of the lateral septum. As established previously, Kiss1 expression in this region is low and regulated by estrogen receptor- and GABAB receptor-dependent mechanisms. Our present experiments on Kiss1-Cre/ZsGreen knock-in mice revealed that transgene expression in the LS begins at Postnatal Day (P)33–36 in females and P40–45 in males and is stimulated by estrogen receptor signaling. Fluorescent cell numbers continue to increase in adulthood and are higher in females. Viral tracing uncovered that the bulk of KPLS fibers joins the medial forebrain bundle and terminates in the hypothalamic supramammillary nucleus. Smaller subsets innervate the medial amygdala or project to other limbic structures. One-quarter of gonadotropin-releasing hormone (GnRH)-immunoreactive perikarya in the preoptic area and their dendrites receive appositions from KPLS axons. OVX adult Kiss1-Cre/ZsGreen mice treated for 4 d with 17β-estradiol or vehicle were used for RNA sequencing studies of laser-microdissected KPLS neurons. The transcriptome included markers of GABAergic and neuropeptidergic (Penk, Cartpt, Vgf) cotransmission and 571 estrogen-regulated transcripts. Estrogen treatment upregulated the acetylcholine receptor transcript Chrm2 and, in slice electrophysiology experiments, caused enhanced muscarinic inhibition of KPLS neurons. Finally, we provided immunohistochemical evidence for homologous neurons in the postmortem human brain, suggesting that KPLS neurons may contribute to evolutionarily conserved regulatory mechanisms. Future studies will need to investigate the putative roles of KPLS neurons in the estrogen-dependent control of GnRH neurons and/or various hypothalamic/limbic functions.