Kisspeptin is a crucial activator of reproductive function, stimulating GnRH neurons in the hypothalamus. However, kisspeptin and its receptor are also expressed in other brain regions including the limbic system, which has key roles in emotional processing. Kisspeptin signalling in the limbic system modulates emotional and sexual brain processing during tasks, however the effects of kisspeptin on underlying resting brain activity have not yet been studied. This is vital for our understanding of reproductive physiology and development of kisspeptin therapeutics. We hypothesised that kisspeptin administration modulates resting brain activity and influences responses to emotional stimuli. To test this, we investigated the effects of kisspeptin administration on brain activity and mood in men. We mapped brain activity using functional MRI in 29 healthy men (mean age 25.0±0.9 years) using a randomised blinded two-way placebo-controlled protocol. The effects of kisspeptin on resting state activity and brain region connectivity were assessed, and correlated to subsequent responses to emotional stimuli and psychometric outcomes. Kisspeptin administration resulted in an increase in circulating kisspeptin (P<0.001) but not testosterone (P=0.180) during the scans, as expected. Kisspeptin enhanced connectivity between key limbic brain structures, including between the hippocampus-caudate (P<0.05) and hippocampus-globus pallidus (P<0.001); structures with established roles in mood regulation and which express kisspeptin receptors. Furthermore, kisspeptins enhancement of resting hippocampus-globus pallidus connectivity predicted increased responses to visual-evoked negative stimuli in several limbic structures (including the thalamus, accumbens, putamen, and cingulate (all P<0.01)). Collectively, these data provide evidence that kisspeptin modulates underlying limbic brain activity and influences subsequent brain responses to negative stimuli. This is the first report of a novel role for kisspeptin in the integration of resting brain activity, negative emotional processing, and reproduction in humans. Therefore, these data have important implications as they suggest that kisspeptin may modulate negative mood with potential therapeutic relevance.