Mammalian GTPase-activating proteins (GAPs) can inhibit innate immunity signaling in a spatiotemporal fashion; however, the role of bacterial GAPs in mediating innate immunity remains unknown. In this study, we show that BspI, a type IV secretion system (T4SS) effector protein, containing a GAP domain at the C terminus, negatively regulates proinflammatory responses and host protection to infection in a mouse model. In macrophages, BspI inhibits the activation of inositol-requiring enzyme 1 (IRE1) kinase, but it does not inhibit activation of ATF6 and PERK. BspI suppresses induction of proinflammatory cytokines via inhibiting the activity of IRE1 kinase caused by VceC, a type IV secretion system effector protein that localizes to the endoplasmic reticulum. Ectopically expressed BspI interacts with IRE1 in HeLa cells. The inhibitory function of BspI depends on its GAP domain but not on interaction with small GTPase Ras-associated binding protein 1B (RAB1B). Collectively, these data support a model where BspI, in a GAP domain-dependent manner, inhibits activation of IRE1 to prevent proinflammatory cytokine responses.