The P2X7 receptor (P2X7R) is a purinoceptor expressed predominantly by cells of immune origin, including microglial cells. P2X7R has a role in the release of biologically active proinflammatory cytokines such as IL-1β, IL-6 and TNFα. Here we demonstrate that when incubated with lipopolysaccharide (LPS), glial cells cultured from brain of P2X7R−/− mice produce less IL-1β compared to glial cells from brains of wild-type mice. This is not the case for TNFα and IL-6. Our results indicate a selective effect of the P2X7R gene deletion on release of IL-1β release but not of IL-6 and TNFα. In addition, we confirm that only microglial cells produce IL-1β, and this release is dependent on P2X7R and ABC1 transporter. Because IL-1β is a key regulator of the brain cytokine network and P2X7R is an absolute requirement for IL-1β release, we further investigated whether response of brain cytokines to LPS in vivo was altered in P2X7R−/− mice compared to wild-type mice. IL-1β and TNFα mRNAs were less elevated in the brain of P2X7R−/− than in the brain of wild-type mice in response to systemic LPS. These results show that P2X7R plays a key role in the brain cytokine response to immune stimuli, which certainly applies also to cytokine-dependent alterations in brain functions including sickness behavior.