During neurite outgrowth, Rho small G protein activity is spatiotemporally regulated to organize the neurite sprouting, extension, and branching. We have previously identified a potent Rho GTPase-activating protein (GAP), RA-RhoGAP, as a direct downstream target of Rap1 small G protein in the neurite outgrowth. In addition to the Ras-associating (RA) domain for Rap1 binding, RA-RhoGAP has the pleckstrin homology (PH) domain for lipid binding. Here, we showed that phosphatidic acid (PA) bound to the PH domain and enhanced GAP activity for Rho. RA-RhoGAP induced extension of neurite in a diacylglycerol kinase-mediated synthesis of the PA-dependent manner. Knockdown of RA-RhoGAP reduced the diacylglycerol kinase-induced neurite extension. In contrast to the effect of the RA domain, the PH domain was specifically involved in the neurite extension, not in the sprouting and branching. These results indicate that PA and Rap1 cooperatively regulate RA-RhoGAP activity for promoting neurite outgrowth.