Nonsteroidal anti-inflammatory drugs (NSAIDs) exert anti-tumor action in a variety of cancer cells. However, several treatment side effects such as gastrointestinal injury, cardiovascular toxicity, and acute renal failure limit their clinical use. We found that indomethacin caused renal epithelial cell injury independently of cyclooxygenase inhibition. Indomethacin treatment was associated with the disruption of mitochondrial transmembrane potential, release of cytochrome c, down-regulation of Bcl-2 and Mcl-1, upregulation of Bax, and elevation of caspases activity. Enhanced Mcl-1 but not Bcl-2 expression alleviated indomethacin-increased caspase-3 activity. Down-regulation of Akt-related and signal transducer and activator of transcription (STAT-3)-related pathways was found in indomethacin-treated cells. Pharmacological and genetic studies revealed a potential mechanistic link between Akt/Mcl-1 and STAT-3/Mcl-1 signaling pathways and indomethacin-induced cytotoxicity. Mcl-1 is a determinant molecule for the induction of epithelial cell injury caused by indomethacin. Therefore, the maintenance of Mcl-1 levels is important for prevention of renal epithelial cell injury and apoptosis.