Acetaminophen (paracetamol) is a widely used analgesic and antipyretic drug with only incompletely understood mechanisms of action. Previous work, using models of acute nociceptive pain, indicated that analgesia by acetaminophen involves an indirect activation of CB₁receptors by the acetaminophen metabolite and endocannabinoid reuptake inhibitor AM 404. However, the contribution of the cannabinoid system to antihyperalgesia against inflammatory pain, the main indication of acetaminophen, and the precise site of the relevant CB₁receptors have remained elusive. Here, we analyzed acetaminophen analgesia in mice of either sex with inflammatory pain and found that acetaminophen exerted a dose-dependent antihyperalgesic action, which was mimicked by intrathecally injected AM 404. Both compounds lost their antihyperalgesic activity inCB₁^−/−mice, confirming the involvement of the cannabinoid system. Consistent with a mechanism downstream of proinflammatory prostaglandin formation, acetaminophen also reversed hyperalgesia induced by intrathecal prostaglandin E₂. To distinguish between a peripheral/spinal and a supraspinal action, we administered acetaminophen and AM 404 tohoxB8-CB₁^−/−mice, which lack CB₁receptors from the peripheral nervous system and the spinal cord. These mice exhibited unchanged antihyperalgesia indicating a supraspinal site of action. Accordingly, local injection of the CB₁receptor antagonist rimonabant into the rostral ventromedial medulla blocked acetaminophen-induced antihyperalgesia, while local rostral ventromedial medulla injection of AM 404 reduced hyperalgesia in wild-type mice but not inCB₁^−/−mice. Our results indicate that the cannabinoid system contributes not only to acetaminophen analgesia against acute pain but also against inflammatory pain, and suggest that the relevant CB₁receptors reside in the rostral ventromedial medulla.SIGNIFICANCE STATEMENTAcetaminophen is a widely used analgesic drug with multiple but only incompletely understood mechanisms of action, including a facilitation of endogenous cannabinoid signaling via one of its metabolites. Our present data indicate that enhanced cannabinoid signaling is also responsible for the analgesic effects of acetaminophen against inflammatory pain. Local injections of the acetaminophen metabolite AM 404 and of cannabinoid receptor antagonists as well as data from tissue-specific CB₁receptor-deficient mice suggest the rostral ventromedial medulla as an important site of the cannabinoid-mediated analgesia by acetaminophen.