Myotubularin-related protein 7 (MTMR7) is a member of the myotubularin (MTM) family. The cDNA encoding the mouse MTMR7 contains 1,983 bp, and the predicted protein has a deduced molecular mass of 75.6 kDa. Northern and Western blot analyses showed that MTMR7 is expressed mainly in brain and mouse neuroblastoma N1E-115 cells. Recombinant MTMR7 dephosphorylated the D-3 position of phosphatidylinositol 3-phosphate and inositol 1,3-bisphosphate [Ins(1,3)P2]. The substrate specificity of MTMR7 is different than other MTM proteins in that this enzyme prefers the water-soluble substrate. Immunofluorescence showed that MTMR7 is localized in Golgi-like granules and cytosol, and subcellular fractionation showed both cytoplasmic and membrane localization of MTMR7 in N1E-115 cells. An MTMR7-binding protein was found in an anti-MTMR7 immunoprecipitate from N1E-115 cells and identified as MTM-related protein 9 (MTMR9) by tandem mass spectrometry. The coiled-coil domain of MTMR9 was sufficient for binding to MTMR7. The binding of MTMR9 increased the Ins(1,3)P2 phosphatase activity of MTMR7. Our results show that MTMR7 forms a complex with MTMR9 and dephosphorylates phosphatidylinositol 3-phosphate and Ins(1,3)P2 in neuronal cells.