When a neurotrophin binds at the presynapse, it sends survival signals all the way to the nucleus on signaling endosomes. These endosomes fuel their own journey with on-board glycolysis—but how is that journey initiated and maintained? Using microfluidic devices and mice, we find that the calcium released upon brain-derived neurotrophic factor (BDNF) binding to its receptor, tropomyosin receptor kinase B (TrkB), is sensed by calcineurin on the cytosolic face of the endosome. Calcineurin dephosphorylates huntingtin, the BDNF scaffold, which sets the endosome moving in a retrograde direction. In an in vitro reconstituted microtubule transport system, controlled calcium uncaging prompts purified vesicles to move to the microtubule minus end. We observed similar retrograde waves of TrkA- and epidermal growth factor receptor (EGFR)-bearing endosomes. Signaling endosomes in neurons thus carry not only their own fuel, but their own navigational system.