Significance G protein-coupled receptors (GPCRs) have long been considered to function primarily at the plasma membrane. Consequently, most drugs are designed to target GPCRs at the cell surface. Ligand-bound GPCRs undergo clathrin- and dynamin-dependent endocytosis. It is uncertain whether GPCRs in endosomes control complex pathophysiological processes in vivo and are a viable therapeutic target. We report that the CGRP receptor signals from endosomes to regulate activity of pain-transmitting neurons in the spinal cord. Lipid-conjugated CGRP receptor antagonists accumulate in endosomes, selectively inhibit endosomal signals, and block sustained excitation of spinal neurons and persistent nociception. The results suggest that GPCRs in endosomes, in addition to those at the cell surface, control ongoing pathophysiological processes in vivo and identify GPCRs in endosomes as a new target for therapy.