Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins (GAPs) for the α-subunit of heterotrimeric G proteins. Several RGS proteins have been found to interact with 14-3-3 proteins. The 14-3-3 protein binding inhibits the GAP function of RGS proteins presumably by blocking their interaction with G α subunit. Since RGS proteins interact with G α subunits through their RGS domains, it is reasonable to assume that the 14-3-3 protein can either sterically occlude the G α interaction surface of RGS domain and/or change its structure. In this work, we investigated whether the 14-3-3 protein binding affects the structure of RGS3 using the time-resolved tryptophan fluorescence spectroscopy. Two single-tryptophan mutants of RGS3 were used to study conformational changes of RGS3 molecule. Our measurements revealed that the 14-3-3 protein binding induces structural changes in both the N-terminal part and the C-terminal RGS domain of phosphorylated RGS3 molecule. Experiments with the isolated RGS domain of RGS3 suggest that this domain alone can, to some extent, interact with the 14-3-3 protein in a phosphorylation-independent manner. In addition, a crystal structure of the RGS domain of RGS3 was solved at 2.3Å resolution. The data obtained from the resolution of the structure of the RGS domain suggest that the 14-3-3 protein-induced conformational change affects the region within the G α -interacting portion of the RGS domain. This can explain the inhibitory effect of the 14-3-3 protein on GAP activity of RGS3.