Relaxin is a polypeptide hormone that triggers multiple signaling pathways through its receptor RXFP1 (relaxin family peptide receptor 1). Many of relaxin's functions, including vascular and antifibrotic effects, are similar to those induced by activation of PPARgamma. In this study, we tested the hypothesis that relaxin signaling through RXFP1 would activate PPARgamma activity. In cells overexpressing RXFP1 (HEK-RXFP1), relaxin increased transcriptional activity through a PPAR response element (PPRE) in a concentration-dependent manner. In cells lacking RXFP1, relaxin had no effect. Relaxin increased both the baseline activity and the response to the PPARgamma agonists rosiglitazone and 15d-PGJ(2), but not to agonists of PPARalpha or PPARdelta. In HEK-RXFP1 cells infected with adenovirus expressing PPARgamma, relaxin increased transcriptional activity through PPRE, and this effect was blocked with an adenovirus expressing a dominant-negative PPARgamma. Knockdown of PPARgamma using siRNA resulted in a decrease in the response to both relaxin and rosiglitazone. Both relaxin and rosiglitazone increased expression of the PPARgamma target genes CD36 and LXRalpha in HEK-RXFP1 and in THP-1 cells naturally expressing RXFP1. Relaxin did not increase PPARgamma mRNA or protein levels. Treatment of cells with GW9662, an inhibitor of PPARgamma ligand binding, effectively blocked rosiglitazone-induced PPARgamma activation, but had no effect on relaxin activation of PPARgamma. These results suggest that relaxin activates PPARgamma activity, and increases the overall response in the presence of PPARgamma agonists. This activation is dependent on the presence of RXFP1. Furthermore, relaxin activates PPARgamma via a ligand-independent mechanism. These studies represent the first report that relaxin can activate the transcriptional activity of PPARgamma.