Phospholipase Cδ3 (PLCδ3) is a key enzyme in phosphoinositide metabolism, however, its physiological function remains unknown. Here we identified the Myosin VI (Myo6) as a binding partner of the PLCδ3. A tail region containing IQ motif and the cargo-binding domain of Myo6, and the C2 domain and PH domain of PLCδ3 were responsible sites for the interaction. Since Myo6 has been well analyzed as one of the "deafness genes" in mouse and human, we examined the expression pattern of PLCδ3 mRNA in the inner ear. In situ hybridization analysis indicated that both Myo6 and PLCδ3 were clearly and limitedly co-expressed in the inner and outer hair cells in the cochlea. Although actin structure of the stereocilia of hair cells seemed to be normal and no detectable hearing defect was observed in PLCδ3 knockout (KO) mice, stable PLCδ3 knockdown in Caco-2 colonic carcinoma cells caused abnormal actin structure of microvilli. In addition, dramatic decrease in expression of Myo6 was observed in intestine of PLCδ3KO mice, where microvilli structure is well developed. These results indicate that PLCδ3 could participate in stability of microvilli structure via regulating and anchoring of Myo6 to plasma membrane.