Neurite outgrowth, a cell differentiation process involving membrane morphological changes, is critical for neuronal network and development. The membrane lipid, phosphatidylinositol (PI) 4,5-bisphosphate (PIP2), is a key regulator of many important cell surface events of membrane signaling, trafficking and dynamics. This lipid is produced mainly by the type I PI 4-phosphate 5-kinase (PIP5K) family members. In this study, we addressed whether PIP5Kα, an isoform of PIP5K, could have a role in neurite outgrowth induced by nerve growth factor (NGF). For this purpose, we knocked down PIP5Kα in PC12 rat pheochromocytoma cells by stable expression of PIP5Kα microRNA that significantly reduced PIP5Kα expression and PIP2 level. Interestingly, NGF-induced neurite outgrowth was more prominent in PIP5Kα-knockdown (KD) cells than in control cells. Conversely, add-back of PIP5Kα into PIP5Kα KD cells abrogated the effect of NGF on neurite outgrowth. NGF treatment activated PI 3-kinase (PI3K)/Akt pathway, which seemed to be associated with reactive oxygen species generation. Similar to the changes in neurite outgrowth, the PI3K/Akt activation by NGF was potentiated by PIP5Kα KD, but was attenuated by the reintroduction of PIP5Kα. Moreover, exogenously applied PIP2 to PIP5Kα KD cells also suppressed Akt activation by NGF. Together, our results suggest that PIP5Kα acts as a negative regulator of NGF-induced neurite outgrowth by inhibiting PI3K/Akt signaling pathway in PC12 cells.