Neurite extension is regulated by multiple signaling cascades that ultimately converge on the actin and microtubule networks . Rho GTPases, molecular switches that oscillate between an inactive, GDP-bound state and an active, GTP-bound state, play a pivotal role in controlling actin cytoskeleton dynamics in the growth cone, whereas the dynamic behavior and interactions of microtubules are largely regulated by proteins called plus-end-tracking proteins (+TIPs), which associate with the ends of growing microtubules. Here, we show that the +TIP Navigator 1 (NAV1) is important for neurite outgrowth and interacts and colocalizes with TRIO, a Rho guanine nucleotide exchange factor that enables neurite outgrowth by activating the Rho GTPases Rac1 and RhoG. We find that binding of NAV1 enhances the affinity of TRIO for Rac1 and RhoG, and that NAV1 regulates TRIO-mediated Rac1 activation and neurite outgrowth. TRIO is also a +TIP, as it interacts with the core +TIP EB1 and tracks microtubule plus ends via EB1 and NAV1. Strikingly, the EB1-mediated recruitment of TRIO to microtubule ends is required for proper neurite outgrowth, and stabilization of the microtubule network by paclitaxel affects both the TRIO-NAV1 interaction and the accumulation of these proteins in neurite extensions. We propose that EB1-labeled ends of dynamic microtubules facilitate the formation and localization of functional NAV1-TRIO complexes, which in turn regulate neurite outgrowth by selectively activating Rac1. Our data reveal a novel link between dynamic microtubules, actin cytoskeleton remodeling, and neurite extension.