Cannabinoid receptors (CB1 and CB2) are promising targets for diverse diseases, such as pain, inflammation, and neurological diseases. However, only a few ligands of CB receptors have reached clinical application so far. In the present study, we focused on the venom of Stephanoconus snails and found an endocannabinoid-like molecule, oleoyl serotonin (OS), active on CB1 and CB2. To determine the effects of OS, CB1 and CB2 receptors were functionally expressed in Xenopus laevis oocytes, together with the G protein-coupled inwardly rectifier potassium channels (GIRK1/2), and a regulator of G protein signaling (RGS4). Ion currents were measured with a two-electrode voltage-clamp system. Results show that OS competitively blocks GIRK1/2 currents induced by WIN55,212-2 (WIN), a non-selective CB agonist, in the CB1-GIRK1/2-RGS4 system. Interestingly, OS acts as a competitive antagonist of CB1 receptors and as a non-competitive blocker of CB2 receptors. To the best of our knowledge, OS is the first venom-derived ligand modulating CB receptors. Furthermore, OS does not show significant activity on serotonin, histamine, opioid and nicotinic receptors, TRP channels or voltage-gated sodium, potassium or calcium channels. To investigate the critical domains of the CB receptor for OS activity, we constructed a chimeric CB1/2 receptor by replacing the transmembrane domain V (TMV)-intracellular loop 3 (ICL3)-TMVI domain (TMV-ICL3-TMVI) of the CB2 receptor with that of the CB1 receptor. Structure-function studies revealed that the TMV-ICL3-TMVI domain of the CB2 receptor is essential for OS to exert its antagonistic effect on CB2 receptors. Furthermore, in vivo evaluation of OS on brain-related neurological functions in mice suggests that OS has an anxiolytic effect, diminishing the stress response in stressful situations. This study highlights the potential of animal venom components as a promising library for the discovery of novel CB receptor ligands.