Cytosolic phospholipase A(2) (cPLA(2)) is the rate-limiting enzyme responsible for the generation of prostaglandins (PGs), which are bioactive lipids that play critical roles in maintaining gastrointestinal (GI) homeostasis. There has been a long-standing association between administration of cyclooxygenase (COX) inhibitors and GI toxicity. GI injury is thought to be induced by suppressed production of GI-protective PGs as well as direct injury to enterocytes. The present study sought to determine how pan-suppression of PG production via a genetic deletion of cPLA(2) impacts the susceptibility to COX inhibitor-induced GI injury. A panel of COX inhibitors including celecoxib, rofecoxib, sulindac, and aspirin were administered via diet to cPLA(2)(-/-) and cPLA(2)(+/+) littermates. Administration of celecoxib, rofecoxib, and sulindac, but not aspirin, resulted in acute lethality (within 2 weeks) in cPLA(2)(-/-) mice, but not in wild-type littermates. Histomorphological analysis revealed severe GI damage following celecoxib exposure associated with acute bacteremia and sepsis. Intestinal PG levels were reduced equivalently in both genotypes following celecoxib exposure, indicating that PG production was not likely responsible for the differential sensitivity. Gene expression profiling in the small intestines of mice identified drug-related changes among a panel of genes including those involved in mitochondrial function in cPLA(2)(-/-) mice. Further analysis of enterocytic mitochondria showed abnormal morphology as well as impaired ATP production in the intestines from celecoxib-exposed cPLA(2)(-/-) mice. Our data demonstrate that cPLA(2) appears to be an important component in conferring protection against COX inhibitor-induced enteropathy, which may be mediated through affects on enterocytic mitochondria.