The medial septum/diagonal band (MS/DB) is a relay region connecting the hypothalamus and brainstem with the hippocampus, and both the MS/DB and dorsal/ventral hippocampus receive strong topographic GABA/peptidergic projections from the nucleus incertus of the pontine tegmentum. The neuropeptide relaxin-3, released by these neurons, is the cognate ligand for a G i/o -protein-coupled receptor, RXFP3, which is highly expressed within the MS/DB, and both cholinergic and GABAergic neurons in this region of rat brain receive relaxin-3 positive terminals/boutons. Comprehensive in vitro studies have demonstrated that the cell signaling pathways altered by RXFP3 stimulation, include inhibition of forskolin-activated cAMP levels and activation of ERK phosphorylation. In this study we investigated whether intracerebroventricular (icv) injection of RXFP3-A2, a selective relaxin-3 receptor agonist, altered ERK phosphorylation levels in the MS/DB of adult male rats. We subsequently assessed the neurochemical phenotype of phosphorylated (p) ERK-positive neurons in MS/DB after icv RXFP3-A2 administration by dual-label immunostaining for pERK and neuronal markers for cholinergic and GABAergic neurons. Central RXFP3-A2 injection significantly increased levels of pERK immunoreactivity (IR) in MS/DB at 20 and 90 min post-injection, compared to vehicle and naive levels. In addition, RXFP3-A2 increased the number of cells expressing pERK-IR in the MS/DB at 90 (but not 20) min post-injection in cholinergic (but not GABAergic) neurons, which also expressed putative RXFP3-IR. Moreover, icv injection of RXFP3-A2 impaired alternation in a delayed spontaneous T-maze test of spatial working memory. The presence of RXFP3-like IR and the RXFP3-related activation of the MAPK/ERK pathway in MS/DB cholinergic neurons identifies them as a key target of ascending relaxin-3 projections with implications for the acute and chronic modulation of cholinergic neuron activity and function by relaxin-3/RXFP3 signaling.