The hypothalamus regulates innate social interactions, but how hypothalamic neurons transduce sex-related sensory signals emitted by conspecifics to trigger appropriate behaviors remains unclear. Here, we addressed this issue by identifying specific hypothalamic neurons required for sensing conspecific male cues relevant to inter-male aggression. By in vivo recording of neuronal activities in behaving mice, we showed that neurons expressing dopamine transporter (DAT+) in the ventral premammillary nucleus (PMv) of the hypothalamus responded to male urine cues in a vomeronasal organ (VNO)-dependent manner in naive males. Retrograde trans-synaptic tracing further revealed a specific group of neurons in the bed nucleus of the stria terminalis (BNST) that convey male-relevant signals from VNO to PMv. Inhibition of PMvDAT+ neurons abolished the preference for male urine cues and reduced inter-male attacks, while activation of these neurons promoted urine marking and aggression. Thus, PMvDAT+ neurons exemplify a hypothalamic node that transforms sex-related chemo-signals into recognition and behaviors. •PMvDAT+ neurons selectively tune to gonad-intact conspecific male urine cues•v-BNST relay male-relevant chemosensory information from VNO to PMvDAT+ neurons•Inhibition of PMvDAT+ neurons blocks male urine preference and decreases attack•Activation of PMvDAT+ neurons promotes urine marking and aggression How hypothalamic neurons transduce sex signals emitted by conspecifics to trigger appropriate behavioral outputs remains unclear. Chen et al. identify PMvDAT+ neurons as a key node that extract male-relevant information from the chemosensory pathway, via the v-BNST, to coordinate multiple behavioral responses, including male urine preference, inter-male aggression, and urine marking.