Background and Purpose Relaxin family peptide receptor 3 (RXFP3) is expressed in brain areas important for processing sensory information and feeding, suggesting that it may be a target for anti-anxiety and anti-obesity drugs. We examined the effects of H3 relaxin, the biased agonist H2 relaxin and the antagonist, R3(B?23-27)R/I5, on RXFP3 signalling to establish their suitability as tools to assess the physiological roles of RXFP3. Experimental Approach The signalling profile of the RXFP3 ligands was determined using reporter gene assays, multiplexed signalling assays and direct examination of receptor-G protein and receptor-?-arrestin interactions using BRET. Key Results H2 relaxin activated p38MAPK and ERK1/2 with lower efficacy than H3 relaxin, but had similar efficacy for JNK1/2 phosphorylation. H2 or H3 relaxin activation of p38MAPK, JNK1/2 or ERK1/2 involved Pertussis toxin-sensitive G-proteins. R3(B?23-27)R/I5 blocked H3 relaxin AP-1 reporter gene activation, but not H2 relaxin AP-1 activation or H3 relaxin NF-?B activation. R3(B?23-27)R/I5 activated the SRE reporter, but did not inhibit either H2 or H3 relaxin SRE activation. R3(B?23-27)R/I5 blocked H3 relaxin-stimulated p38MAPK and ERK1/2 phosphorylation, but was a weak partial agonist for p38MAPK and ERK1/2 signalling. p38MAPK activation by R3(B?23-27)R/I5 was G protein-independent. H3 relaxin-activated RXFP3 interacts with Gai2, Gai3, GaoA and GaoB whereas H2 relaxin or R3(B?23-27)R/I5 induce interactions only with Gai2 or GaoB. Only H3 relaxin promoted RXFP3/?-arrestin interactions that were blocked by R3(B?23-27)R/I5. Conclusion and Implications Understanding signalling profile of drugs acting at RXFP3 is essential for development of therapies targeting this receptor.