Relaxin-3 is a conserved neuropeptide expressed by neurons in the brainstem nucleus incertus (NI) and periaqueductal gray (PAG), pontine raphe (PnR), and dorsal to substantia nigra (dSN). Ascending relaxin-3 projection patterns to receptor (RXFP3)-rich areas suggest a role in arousal- and stress-related functions including feeding and anxiety1,2. In rats, intracerebroventricular (icv) and intra-PVN infusion of relaxin-3 increased HPA axis activity and feeding3,4, while icv injection of an RXFP3 agonist (RXFP3-A2) reduced anxiety- and depressive-like behaviours5. Notably, similar studies in mice have not been completed. Initial anatomical studies assessed the number of relaxin-3-positive neurons in mouse brain: ~435 in NI, ~70 in PnR, ~115 in PAG, and ~200 in dSN. In functional studies, mice given central injections of RXFP3-A2 (iPVN, 0.1 nmol; icv, 1 nmol), displayed no significant increase in 1-h food intake (iPVN, p=0.63; icv, p=0.49), or altered HPA axis activity (serum corticosterone, iPVN, p=0.33; icv, p=0.55). An RXFP3 antagonist (R3B(1-22)R, 0.4 nmol, icv) did not attenuate serum corticosterone (p=0.89), but reduced food anticipatory activity (p=0.02) and palatable- (p=0.04), post-mild food deprivation- (p<0.001), and dark phase- (p=0.03) food consumption. Effects of RXFP3 activation (RXFP3-A2, 1 nmol, icv) on anxiety behaviour were investigated in multiple tests. No significant differences were observed in time spent in aversive areas, an indicator of anxiety-like behaviour, in the elevated plus maze (p=0.12), large open-field (p=0.84), light/dark box (p=0.45), or social avoidance (p=0.24) tests. However, in the three-chamber social preference test, RXFP3-A2 reduced anxiety-like behaviour, reflected by an increased total interaction with novel mice, compared to vehicle (p=0.04), in mice displaying high, but not low, anxiety. These studies suggest relaxin-3/RXFP3 exerts modulatory effects during high, but not basal, arousal states in mice. Differences between rats and mice may arise from differential RXFP3 expression and/or downstream coupling, and will be further investigated using RXFP3-Cre/YFP reporter mice.