Although several non-receptor activators of heterotrimeric G proteins have been identified, the structural features of G proteins that determine their interaction with such activators and the subsequent biological effects are poorly understood. Here we investigated the structural determinants in G alpha(i3) necessary for its regulation by GIV/girdin, a guanine-nucleotide exchange factor (GEF) that activates G alpha(i) subunits. Using G protein activity and in vitro pulldown assays we demonstrate that G alpha(i3) is a better substrate for GIV than the highly homologous G alpha(o). We identified Trp-258 in the G alpha(i) subunit as a novel structural determinant for GIV binding by comparing GIV binding to G alpha(i3)/G alpha(o) chimeras. Mutation of Trp-258 to the corresponding Phe in G alpha(o) decreased GIV binding in vitro and in cultured cells but did not perturb interaction with other G alpha-binding partners, i.e. G betagamma, AGS3 (a guanine nucleotide dissociation inhibitor), GAIP/RGS19 (a GTPase-activating protein), and LPAR1 (a G protein-coupled receptor). Activation of G alpha(i3) by GIV was also dramatically reduced when Trp-258 was replaced with Tyr, Leu, Ser, His, Asp, or Ala, highlighting that Trp is required for maximal activation. Moreover, when mutant G alpha(i3) W258F was expressed in HeLa cells they failed to undergo cell migration and to enhance Akt signaling after growth factor or G protein-coupled receptor stimulation. Thus activation of G alpha(i3) by GIV is essential for biological functions associated with G alpha(i3) activation. In conclusion, we have discovered a novel structural determinant on G alpha(i) that plays a key role in defining the selectivity and efficiency of the GEF activity of GIV on G alpha(i) and that represents an attractive target site for designing small molecules to disrupt the G alpha(i)-GIV interface for therapeutic purposes.