Abstract What We Already Know about This Topic What This Article Tells Us That Is New Background Pyroptosis, a type of proinflammatory programmed cell death, drives cytokine storm. Caspase-11–dependent macrophage pyroptosis contributes to mortality during sepsis. Sphingosine-1-phosphate receptor 2 (S1PR2) signaling can amplify interleukin-1β secretion in endotoxin-induced inflammation. Here, we hypothesized that S1PR2 signaling increases caspase-11–dependent macrophage pyroptosis and worsens Gram-negative sepsis outcome. Methods A Gram-negative sepsis model was induced through intraperitoneal injection of Escherichia coli. Primary peritoneal macrophages isolated from wild-type, S1pr2-deficient (S1pr2 -/-), or nucleotide-binding oligomerization domain-like receptor protein-3–deficient mice were treated with E. coli. Caspase-11 activation, macrophage pyroptosis, and Ras homolog gene family, member A-guanosine triphosphate levels were assessed in those cells. Additionally, monocyte caspase-4 (an analog of caspase-11) expression and its correlation with S1PR2 expression were determined in patients with Gram-negative sepsis (n = 11). Results Genetic deficiency of S1PR2 significantly improved survival rate (2/10 [20%] in wild-type vs. 7/10 [70%] in S1pr2 -/-, P = 0.004) and decreased peritoneal macrophage pyroptosis (pyroptosis rate: 35 ± 3% in wild-type vs. 10 ± 3% in S1pr2 -/-, P < 0.001). Decreased caspase-11 activation in S1PR2 deficiency cells contributed to the reduced macrophage pyroptosis. In addition, RhoA inhibitor abrogated the amplified caspase-11 activation in wild-type or S1PR2-overexpressing cells. In patients with Gram-negative sepsis, caspase-4 increased significantly in monocytes compared to nonseptic controls and was positively correlated with S1PR2 (r = 0.636, P = 0.035). Conclusions S1PR2 deficiency decreased macrophage pyroptosis and improved survival in E. coli sepsis. These beneficial effects were attributed to the decreased caspase-11 activation of S1PR2-deficient macrophages. S1PR2 and caspase-11 may be promising new targets for treatment of sepsis.