Small intestinal ulceration, bleeding, and inflammation are major adverse effects associated with the use of diclofenac (DCF) or other nonsteroidal anti-inflammatory drugs (NSAIDs). The underlying mechanisms of DCF enteropathy are poorly understood, but there is increasing evidence that topical effects are involved. The aim of this study was to explore the role of c-Jun-N-terminal kinase (JNK) in DCF-induced enterocyte death because JNK not only regulates mitochondria-mediated apoptosis but also is a key node where many of the proximal stress signals converge. Male C57BL/6J mice were injected intraperitoneally with DCF or vehicle (Solutol HS-15), and the extent of small intestinal ulceration was determined. A single dose of DCF (60 mg/kg) produced numerous ulcers in the third and fourth quartiles of the jejunum and ileum, with maximal effects after 18 h and extensive recovery after 48 h. To study the molecular pathways leading to enterocyte injury, we isolated villi-enriched mucosal fractions from DCF-treated mice. Immunoblot studies with a phosphospecific JNK antibody revealed that JNK1/2 (p46) was activated at 6 h, leading to phosphorylation of the downstream target c-Jun. The levels of the JNK-regulated proapoptotic transcription factor C/EBP homologous protein (CHOP) were also increased after DCF. The selective JNK inhibitor SP600125 (30 mg/kg ip), given both 1 h before and 1 h after DCF, blocked JNK kinase activity and afforded significant protection against DCF enteropathy. In conclusion, these data demonstrate that the JNK pathway is critically involved in the pathogenesis of DCF-induced enteropathy and suggest a potential application of JNK inhibitors in the prevention of NSAID-induced enteropathy.