Cannabinoid receptor 1 (CB1) is the principal target of Δ9-tetrahydrocannabinol (THC), a psychoactive chemical from Cannabis sativa with a wide range of therapeutic applications and a long history of recreational use. CB1 is activated by endocannabinoids and is a promising therapeutic target for pain management, inflammation, obesity, and substance abuse disorders. Here, we present the 2.8 Å crystal structure of human CB1 in complex with AM6538, a stabilizing antagonist, synthesized and characterized for this structural study. The structure of the CB1-AM6538 complex reveals key features of the receptor and critical interactions for antagonist binding. In combination with functional studies and molecular modeling, the structure provides insight into the binding mode of naturally occurring CB1 ligands, such as THC, and synthetic cannabinoids. This enhances our understanding of the molecular basis for the physiological functions of CB1 and provides new opportunities for the design of next-generation CB1-targeting pharmaceuticals. Display omitted •AM6538 is presented as a stabilizing, tight binding antagonist of CB1•Crystal structure of human CB1 in complex with AM6538 is determined•Molecular docking predicts CB1 binding modes of THC and synthetic cannabinoids•Resolution of the binding pocket provides path for rational CB1 drug design A look at ligand engagement with the human CB1 receptor opens up avenues for design of new modulators with favorable physiological profiles.