P2X7 receptor plays important roles in inflammation and immunity, and thereby it serves as a potential therapeutic target for inflammatory diseases. Rhein, an anthraquinone derivative, exhibits significant anti-inflammatory and immunosuppressive activities in therapy. However, the underlying mechanisms are largely unclear. Here, we aimed to investigate the effects of rhein on P2X7 receptor-mediated responses in vitro. In HEK293 cells expressing rat P2X7 receptor, we first found that rhein concentration-dependently blocked ATP-induced cytosolic calcium concentration ([Ca(2+)]c) elevation and pore formation of the plasma membrane, two hallmarks of the P2X7 receptor activation. These two inhibitory effects of rhein were also observed in rat peritoneal macrophages. Furthermore, rhein counteracted macrophage phagocytosis attenuation and suppressed reactive oxygen species (ROS) production triggered by ATP/BzATP. Meanwhile, rhein reduced ATP/BzATP-induced IL-1β release in lipopolysaccharide-activated macrophages. Prolonged application of ATP caused macrophage apoptosis, while the presence of rhein suppressed this cell cytotoxicity. Such ATP/BzATP-induced cellular reactions were also inhibited by a well-known rat P2X7 receptor antagonist, brilliant blue G, in a similar way to rhein. Together, our results demonstrate that rhein inhibit ATP/BzATP-induced [Ca(2+)]c increase, pore formation, ROS production, phagocytosis attenuation, IL-1β release and cell apoptosis by antagonizing the P2X7 receptor in rat peritoneal macrophages.