Nogo-A is one of the most potent myelin-associated inhibitors for axonal growth, regeneration, and plasticity in the adult central nervous system. The Nogo-A–specific fragment NogoΔ20 induces growth cone collapse, and inhibits neurite outgrowth and cell spreading by activating RhoA. Here, we show that NogoΔ20 is internalized into neuronal cells by a Pincher- and rac-dependent, but clathrin- and dynamin-independent, mechanism. Pincher-mediated macroendocytosis results in the formation of NogoΔ20-containing signalosomes that direct RhoA activation and growth cone collapse. In compartmentalized chamber cultures, NogoΔ20 is endocytosed into neurites and retrogradely transported to the cell bodies of dorsal root ganglion neurons, triggering RhoA activation en route and decreasing phosphorylated cAMP response element binding levels in cell bodies. Thus, Pincher-dependent macroendocytosis leads to the formation of Nogo-A signaling endosomes, which act both within growth cones and after retrograde transport in the cell body to negatively regulate the neuronal growth program.