We previously showed that group II phospholipase A₂ (PLA₂-II), a secretory, bactericidal, and proinflammatory protein in intestinal crypts, is upregulated after lipopolysaccharide (LPS) and platelet-activating factor (PAF) challenge. Here we examined whether germ-free environment (GF) or antibiotic treatment (ABX) affects the pathophysiological responses and intestinal PLA₂-II activity after PAF (1.5 µg/kg) or LPS (8 mg/kg) injection. We found that LPS and PAF induced hypotension and mild intestinal injury in conventionally fed (CN) rats; these changes were milder in ABX rats, whereas GF rats showed no intestinal injury. PLA₂-II enzyme activity was detected in normal rat small intestine; the basal level was not diminished in ABX or GF rats. PAF and LPS caused an increase in PLA₂-II activity, which was abrogated in GF and ABX rats. Recolonization of GF rats by enteral contamination restituted their PLA₂-II response to PAF and LPS and susceptibility to bowel injury. We conclude that PAF- and LPS-induced increases in PLA₂-II activity are dependent on gut bacteria, and ABX and GF rats are less susceptible to LPS-induced injury than CN rats.