The lateral septum (LS) integrates diverse subcortical and cortical inputs to generate appropriate affective behavioral responses related to fear, reward, social behaviors, and memory. Contrary to the medial septum (MS) the LS is considered an entirely GABAergic nucleus where neurons express a plethora of GABAergic markers in a partially overlapping manner. In this work, we describe a neglected and underappreciated neuronal subclass in the lateral septum expressing choline-acetyltransferase (ChAT), therefore named Lateral Septum Cholinergic Neurons (LSCNs). ChAT expressing neurons are immunohistochemically detectable in mouse, macaque, and post-mortem human LS sections, discontinuous of MS cholinergic cells with smaller soma diameter measured in all three species. Furthermore, LSCNs can be selectively targeted in multiple independent transgenic mouse lines. Using AAV-mediated anterograde, and ΔRabies mediated monosynaptic retrograde tracings, we reveal that LSCNs integrate inputs from the hippocampus, hypothalamic areas, and the basal forebrain, furthermore project robustly to the hypothalamus and the medial amygdala. In vitro electrophysiological recordings of hypothalamic neurons show acetylcholine release from selectively stimulated LSCN axons. To better understand their function, we parallelly recorded LSCN activity (GCaMP8) and their ACh release (ACh3.0) during a head-restrained probabilistic Pavlovian conditioning task and freely moving behaviors via fiber photometry. Optical recordings revealed that LSCNs are activated by different types of aversive stimuli (air puff, foot shock), but respond robustly to reward and social stimuli as well, which responses are paralleled by ACh release in LSCN target regions. Further experiments are aimed at understanding their functional significance.