||Heterotrimeric G proteins regulate diverse physiological processes by modulating the activities of intracellular effectors. Members of the Gαq family link activation of G protein-coupled receptors (GPCRs) to the stimulation of phospholipase Cβ (PLCβ) and calcium mobilization. However, they differ markedly in biochemical properties as well as tissue distribution. Recent findings also showed that some of the cellular activities of Gαq family members are independent of PLCβ activation. Novel binding partners of Gαq subunits have also been reported. However, little is known about proteins that interact with other members of the Gαq family, such as the hematopoietic specific Gα16. The research aim of this study is to identify Gα16-interacting proteins and the roles of protein complexes in Gα16-mediated signaling. A guanine nucleotide exchange factor, p63RhoGEF, has been shown to interact with Gαq/11 proteins and thus provides linkage to RhoA activation. 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 Gα16 (Gα16QL). Interestingly, overexpression of p63RhoGEF inhibited Gα16QL-induced IP3 production in a concentration-dependent manner. The binding of PLCβ2 to Gα16QL could be displaced by p63RhoGEF. Similarly, p63RhoGEF inhibited the binding of tetratricopeptide repeat 1 to Gα16QL, leading to a suppression of Gα16QL-induced Ras activation. In the presence of p63RhoGEF, Gα16QL-induced STAT3 phosphorylation was significantly reduced and Gα16QL-mediated SRE transcriptional activation was attenuated. In addition, it was found that multiple regions of Gα16 are responsible for binding with p63RhoGEF. Taken together, these results suggest that p63RhoGEF binds to activated Gα16 and inhibits its signaling pathways. Apart from p63RhoGEF, our co-immunoprecipitation assay also suggested that Gα16 could interact with class IA PI3Ks but not class IB PI3K. In contrast to the observation in Gα16/p63RhoGEF complex; both Gα16 and Gα16QL can effectively form signaling complexes with class IA PI3Ks. Differential characteristics between Gα16 and Gαq were observed in terms of the selectivity of PI3K isoforms. Gα16 bound to both p110α and p110β but not p110δ. Signaling complexes of Gα16/p110α could be found in hematopoietic cells which endogenously express Gα16. It was also found that overexpression of class IA PI3Ks did not affect Gα16QL-mediated PLC activation. In addition, the immunoprecipitation assay showed that overexpression of p85/p110α or p85/p110β did not interfere with the binding with Gα16 and PLCβ2. Furthermore, Gα16QL attenuated PI3K-induced Akt phosphorylation and PIP3 level. Collectively, the present study provides evidence that Gα16 interacts with signaling molecules other than PLCβ and modulates different signaling pathways.