G-protein signaling modulator-3 (GPSM3), also known as G18 or AGS4, is a member of the Gαi/o-Loco (GoLoco) motif containing proteins.GPSM3acts through its two GoLoco motifs to exert GDP dissociation inhibitor activity over Gαi subunits; recently revealed is the existence of an additional regulatory site within GPSM3 directed toward monomeric Gβ subunits during their biosynthesis. Here, using in silico and proteomic approaches, we have found that GPSM3 also interacts directly with numerous members of the 14-3-3 protein family. This interaction is dependent onGPSM3phosphorylation, creating a mode II consensus 14-3-3 binding site. 14-3-3 binding to the N-terminal disordered region of GPSM3 confers stabilization from protein degradation. The complex of GPSM3 and 14-3-3 is exclusively cytoplasmic, and both moieties mutually control their exclusion from the nucleus. Phosphorylation of GPSM3 by a proline-directed serine/threonine kinase and the resultant association of 14-3-3 is the first description of post-translational regulation of GPSM3 subcellular localization, a process that likely regulates important spatio-temporal aspects of G-protein-coupled receptor signaling modulation by GPSM3.