Interleukin-1β (IL-1β) is involved in mood alterations associated with inflammatory illnesses and with stress. The synaptic basis of IL-1β-induced emotional disturbances is still unknown. To address the possible involvement of the endocannabinoid system in IL-1β-induced anxiety, we performed behavioral and neurophysiological studies in mice exposed to stress or to intracerebroventricular injections of this inflammatory cytokine or of its antagonist. We found that a single intracerebroventricular injection of IL-1β caused anxiety in mice, and abrogated the sensitivity of cannabinoid CB1 receptors (CB1Rs) controlling GABA synapses in the striatum. Identical behavioral and synaptic results were obtained following social defeat stress, and intracerebroventricular injection of IL-1 receptor antagonist reverted both effects. IL-1β-mediated inhibition of CB1R function was secondary to altered cholesterol composition within membrane lipid rafts, and required intact function of the transient receptor potential vanilloid 1 (TRPV1) channel, another element of the endocannabinoid system. Membrane lipid raft disruption and inhibition of cholesterol synthesis, in fact, abrogated IL-1β-CB1R coupling, and TRPV1-/- mice were indeed insensitive to the synaptic and behavioral effects of both IL-1β and stress. On the other hand, cholesterol enrichment of striatal slices mimicked the synaptic effects of IL-1β on CB1Rs only in control mice, while the same treatment was ineffective in slices prepared from TRPV1-/- mice. The present investigation identifies a previously unrecognized interaction between a major proinflammatory cytokine and the endocannabinoid system in the pathophysiology of anxiety.