Adhesion G protein-coupled receptors (aGPCR) are characterized by a large extracellular region containing a conserved GPCR-autoproteolysis-inducing (GAIN) domain. Despite their relevance to several disease conditions, we do not understand the molecular mechanism by which aGPCRs are physiologically activated. GPR110 (ADGRF1) was recently deorphanized as the functional receptor of N-docosahexaenoylethanolamine (synaptamide), a potent synaptogenic metabolite of docosahexaenoic acid. Thus far, synaptamide is the first and only small-molecule endogenous ligand of an aGPCR. Here, we demonstrate the molecular basis of synaptamide-induced activation of GPR110 in living cells. Using in-cell chemical cross-linking/mass spectrometry, computational modeling and mutagenesis-assisted functional assays, we discover that synaptamide specifically binds to the interface of GPR110 GAIN subdomains through interactions with residues Q511, N512 and Y513, causing an intracellular conformational change near TM6 that triggers downstream signaling. This ligand-induced GAIN-targeted activation mechanism provides a framework for understanding the physiological function of aGPCRs and therapeutic targeting in the GAIN domain.