Objective: Ceramide is a key player in the induction of pancreatic β-cell apoptosis, insulin resistance, and reduction of insulin gene expression. Thioredoxin-Interacting Protein (TXNIP) has also emerged as an important regulator in β-cell biology and diabetes development. Elevated TXNIP expression by high glucose and diabetes induced β-cell apoptosis whereas inhibition or knock-out of TXNIP protected β-cells dysfunction. However, the effects of ceramides on TXNIP expressions have remained largely unknown. To identify the mediator of ceramide in pancreatic β-cell dysfunction, we investigated the expression and pathophysiological role of TXNIP in response to ceramide to induce β-cell dysfunction and apoptosis in insulin producing cells. Methods: INS-cells and primary rat islets were treated with ceramide and we measured TXNIP mRNA and protein expression. In addition, we also characterized the signaling pathways in response to ceramide. Results: Ceramide treatment increased β-cell apoptosis and TXNIP expression as well as down regulated Insulin and PDX-1 expression. Moreover, ceramide treatment increased mitochondrial membrane potential loss resulting in cytochrome c release which leads to caspase-3 activation. Prolonged exposure to ceramide also activated several MAPKs, including p38 MAPK and JNK. Intriguingly, TXNIP knockdown attenuated INS-1 cell apoptosis, caspase-3 activation, reactive oxygen species production and morphological alterations due to ceramide. Conclusion: Our data demonstrated that TXNIP might play an important role in the ceramide induced pathogenesis of β-cell damage and serving as a potential molecular target in the modulation of pancreatic beta cell dysfunction and failure.