Heterotrimeric G-proteins bind to cell-surface receptors and are integral in transmission of signals from outside the cell. Upon activation of the Gα subunit by binding of GTP, the Gα and Gβγ subunits dissociate and interact with effector proteins for signal transduction. Regulatory proteins with the 19-amino-acid GoLoco motif can bind to Gα subunits and maintain G-protein subunit dissociation in the absence of Gα activation. Here we describe the structural determinants of GoLoco activity as revealed by the crystal structure of Gαi1-GDP bound to the GoLoco region of the 'regulator of G-protein signalling' protein RGS14. Key contacts are described between the GoLoco motif and Gα protein, including the extension of GoLoco's highly conserved Asp/Glu-Gln-Arg triad into the nucleotide-binding pocket of Gα to make direct contact with the GDP α- and β-phosphates. The structural organization of the GoLoco-Gαi1 complex, when combined with supporting data from domain-swapping experiments, suggests that the Gα all-helical domain and GoLocoregion carboxy-terminal residues control the specificity of GoLoco-Gα interactions.