OBJECTIVE - We have previously shown that lack of thioredoxin-interacting protein (TXNIP) protects against diabetes and glucotoxicity-induced β-cell apoptosis. Because the role of TXNIP in lipotoxicity is unknown, the goal of the present study was to determine whether TXNIP expression is regulated by fatty acids and whether TXNIP deficiency also protects β-cells against lipoapoptosis. RESARCH DESIGN AND METHODS - To determine the effects of fatty acids on β-cell TXNIP expression, INS-1 cells and isolated islets were incubated with/without palmitate and rats underwent cyclic infusions of glucose and/or Intralipid prior to islet isolation and analysis by quantitative real-time RT-PCR and immunoblotting. Using primary wild-type and TXNIP-deficient islets, we then assessed the effects of palmitate on apoptosis (transferase-mediated dUTP nick-end labeling [TUNEL]), mitochondrial death pathway (cytochrome c release), and endoplasmic reticulum (ER) stress (binding protein [BiP], C/EBP homologous protein [CHOP]). Effects of TXNIP deficiency were also tested in the context of staurosporine (mitochondrial damage) or thapsigargin (ER stress). RESULTS - Glucose elicited a dramatic increase in islet TXNIP expression both in vitro and in vivo, whereas fatty acids had no such effect and, when combined with glucose, even abolished the glucose effect. We also found that TXNIP deficiency does not effectively protect against palmitate or thapsigargin-induced β-cell apoptosis, but specifically prevents staurosporine- or glucose-induced toxicity. CONCLUSIONS - Our results demonstrate that unlike glucose, fatty acids do not induce β-cell expression of proapoptotic TXNIP. They further reveal that TXNIP deficiency specifically inhibits the mitochondrial death pathway underlying β-cell glucotoxicity, whereas it has very few protective effects against ER stress-mediated lipoapoptosis. © 2010 by the American Diabetes Association.