Abstract Human caspase-4 and its mouse homolog caspase-11 are receptors for cytoplasmic lipopolysaccharide. Activation of the caspase-4/11-dependent NLRP3 inflammasome is required for innate defense and endotoxic shock, but how caspase-4/11 is modulated remains unclear. Here, we show that mice lacking the oxidative stress sensor glutathione peroxidase 8 (GPx8) are more susceptible to colitis and endotoxic shock, and exhibit reduced richness and diversity of the gut microbiome. C57BL/6 mice that underwent adoptive cell transfer of GPx8-deficient macrophages displayed a similar phenotype of enhanced colitis, indicating a critical role of GPx8 in macrophages. GPx8 bound covalently to caspase-4/11 via disulfide bonding between cysteine 79 of GPx8 and cysteine 118 of caspase-4, which restrained caspase-4/11 activation, while GPx8 deficiency led to caspase-4/11-induced inflammation during colitis and septic shock. Inhibition of caspase-4/11 activation with small molecules reduced the severity of colitis in GPx8-deficient mice. Notably, colonic tissue from patients with ulcerative colitis displayed low levels of Gpx8 and high caspase-4 expression. In conclusion, these results suggest that GPx8 protects against colitis by negatively regulating caspase-4/11 activity.