Heterotrimeric G proteins regulate diverse physiological processes by modulating the activities of intracellular effectors. Members of the Galpha(q) family link G protein-coupled receptor activation to phospholipase Cbeta (PLCbeta) activity and intracellular calcium signaling cascades. However, they differ markedly in biochemical properties as well as tissue distribution. Recent findings have shown that some of the cellular activities of Galpha(q) family members are independent of PLCbeta activation. A guanine nucleotide exchange factor, p63RhoGEF, has been shown to interact with Galpha(q) proteins and thus provides linkage to RhoA activation. However, it is not known if p63RhoGEF can associate with other Galpha(q) family members such as Galpha(16). In the present study, we employed co-immunoprecipitation studies in HEK293 cells to demonstrate that p63RhoGEF can form a stable complex with the constitutively active mutant of Galpha(16) (Galpha(16)QL). Interestingly, overexpression of p63RhoGEF inhibited Galpha(16)QL-induced IP(3) production in a concentration-dependent manner. The binding of PLCbeta(2) to Galpha(16)QL could be displaced by p63RhoGEF. Similarly, p63RhoGEF inhibited the binding of tetratricopeptide repeat 1 to Galpha(16)QL, leading to a suppression of Galpha(16)QL-induced Ras activation. In the presence of p63RhoGEF, Galpha(16)QL-induced STAT3 phosphorylation was significantly reduced and Galpha(16)QL-mediated SRE transcriptional activation was attenuated. Taken together, these results suggest that p63RhoGEF binds to activated Galpha(16) and inhibits its signaling pathways.