Nonsteroidal anti-inflammatory drugs (NSAIDs) are used extensively as therapeutic agents, despite their well documented gastrointestinal (GI) toxicity. At this time, the mechanisms responsible for NSAID-associated GI damage are incompletely understood. In this study, we used microarray analysis to generate a novel hypothesis about cellular mechanisms that underlie the GI toxicity of NSAIDs. Monolayers of intestinal epithelial cells (IEC-6) were treated with NSAIDs that either exhibit (indomethacin, NS-398 [N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide]) or lack (SC-560 [5-(4-chlorphenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole]) inhibitory effects on IEC-6 migration. Bioinformatic analysis of array data identified the calpain cysteine proteases and their endogenous inhibitor calpastatin as potential targets of NSAIDs shown previously to retard IEC-6 migration. Accordingly, quantitative real-time reverse transcription polymerase chain reaction and immunoblotting were performed to assess the effects of NSAIDs on the expression of mRNA and protein for calpain 8, calpain 2, calpain 1, and calpastatin. In treated IEC-6 monolayers, NS-398 decreased the expression of mRNA for calpain 2 and calpain 8. Both NS-398 and indomethacin decreased the protein expression of calpains 8, 2, and 1. None of the NSAIDs affected expression of calpastatin mRNA or protein. The calpain inhibitors, N-acetyl-Leu-Leu-methioninal and N-acetyl-Leu-Leu-Nle-CHO, retarded IEC-6 cell migration in a concentration-dependant fashion, and these inhibitory effects were additive with those of indomethacin and NS-398. Our experimental results suggest that the altered expression of calpain proteins may contribute to the adverse effects of NSAIDs on intestinal epithelial restitution.