Phosphatidylinositol 4-phosphate 5-kinase (PIP-5kin) regulates actin cytoskeletal reorganization through its product phosphatidylinositol 4,5-bisphosphate. In the present study we demonstrate that PIP-5kin is essential for neurite remodeling, which is regulated by actin cytoskeletal reorganization in neuroblastoma N1E-115 cells. Overexpression of wild-type mouse PIP-5kin-alpha inhibits the neurite formation that is normally stimulated by serum depletion, whereas a lipid kinase-defective mutant of PIP-5kin-alpha, D266A, triggers neurite extension even in the presence of serum and blocks lysophosphatidic acid-induced neurite retraction. These results phenocopy those previously reported for the small GTPase RhoA and its effector p160 Rho-associated coiled coil-forming protein kinase (ROCK). However, the ROCK-specific inhibitor Y-27632 failed to block the inhibition by PIP-5kin-alpha of neurite extension, whereas D266A did block the neurite retraction induced by overexpression of ROCK. These results, taken together, suggest that PIP-5kin-alpha functions as a downstream effector for RhoA/ROCK to couple lysophosphatidic acid signaling to neurite retraction presumably through its product phosphatidylinositol 4,5-bisphosphate.