Class II phosphoinositide 3-kinases (PI3Ks), PI3K-C2α and PI3K-C2β, are highly homologous and distinct from class I and class III PI3Ks in catalytic products and domain structures. In contrast to class I and class III PI3Ks, physiological roles of PI3K-C2α and PI3K-C2β are not fully understood. Because we previously demonstrated that PI3K-C2α is involved in vascular smooth muscle contraction, we studied the phenotypes of smooth muscle-specific knockout (KO) mice of PI3K-C2α and PI3K-C2β. The pup numbers born from single PI3K-C2α-KO and single PI3K-C2β-KO mothers were similar to those of control mothers, but those from double KO (DKO) mothers were smaller compared with control mice. However, the number of intrauterine fetuses in pregnant DKO mothers was similar to that in control mice. Both spontaneous and oxytocin-induced contraction of isolated uterine smooth muscle (USM) strips was diminished in DKO mice but not in either of the single KO mice, compared with control mice. Furthermore, contraction of USM of DKO mice was less sensitive to a Rho kinase inhibitor. Mechanistically, the extent of oxytocin-induced myosin light chain phosphorylation was greatly reduced in USM from DKO mice compared with control mice. The oxytocin-induced rise in the intracellular Ca2+ concentration in USM was similar in DKO and control mice. However, Rho activation in the intracellular compartment was substantially attenuated in DKO mice compared with control mice, as evaluated by fluorescence resonance energy transfer imaging technique. These data indicate that both PI3K-C2α and PI3K-C2β are required for normal USM contraction and parturition mainly through their involvement in Rho activation.