Phosphatidylinositol 3-kinase (PI3K) mediates receptor tyrosine kinase and G protein coupled receptor (GPCR) signaling by phosphorylating phosphoinositides to elicit various biological responses. Galpha(q) has previously been shown to inhibit class IA PI3K by interacting with the p110alpha subunit. However, it is not known if PI3Ks can associate with other Galpha(q) family members such as Galpha(16). Here, we demonstrated that class IA PI3Ks, p85/p110alpha and p85/p110beta, could form stable complexes with wild type Galpha(16) and its constitutively active mutant (Galpha(16)QL) in HEK293 cells. In contrast, no interaction between Galpha(16) and class IB PI3K was observed. The Galpha(16)/p110alpha signaling complex could be detected in hematopoietic cells that endogenously express Galpha(16). Overexpression of class I PI3Ks did not inhibit Galpha(16)QL-induced IP(3) production and, unlike p63RhoGEF, class IA PI3Ks did not attenuate the binding of PLCbeta(2) to Galpha(16)QL. On the contrary, the function of class IA PI3Ks was suppressed by Galpha(16)QL as revealed by diminished production of PIP(3) as well as inhibition of EGF-induced Akt phosphorylation. Taken together, these results suggest that Galpha(16) can bind to class IA PI3Ks and inhibit the PI3K signaling pathway.