Heterotrimeric G proteins of the G12 family regulate the Rho GTPase through RhoGEFs that contain an amino-terminal regulator of G protein signaling (RGS) domain (RGS-RhoGEFs). Direct regulation of the activity of RGS-RhoGEFs p115 or leukemia-associated RhoGEF (LARG) by Galpha13 has previously been demonstrated. However, the precise biochemical mechanism by which Galpha13 stimulates the RhoGEF activity of these proteins has not yet been well understood. Based on the crystal structure of Galphai1 in complex with RGS4, we mutated the Galpha13 residue lysine 204 to alanine (Galpha13K204A) and characterized the effect of this mutation in its regulation of RGS-RhoGEFs p115 or LARG. Compared with wild-type Galpha13, Galpha13K204A induced much less serum-response factor activation when expressed in HeLa cells. Recombinant Galpha13K204A exhibits normal function in terms of nucleotide binding, basal GTP hydrolysis, and formation of heterotrimer with betagamma. We found that lysine 204 of Galpha13 is important for interaction with the RGS domain of p115 or LARG and for the GTPase-activating protein activity of these proteins. In addition, the K204A mutation of Galpha13 impaired its regulation of the RhoGEF activity of p115 or LARG. We conclude that lysine 204 of Galpha13 is important for interaction with RGS-RhoGEFs and is critically involved in the regulation of their activity.