Insulin resistance is a key condition in the development of type 2 diabetes. It is well established that exacerbated Jun NH2-terminal kinase (JNK) activity is involved in promoting insulin resistance in peripheral insulin-target tissues; however, this involvement is less documented in pancreatic beta-cells. Using a transgenic mouse model, here we show that JNK activation in beta-cells led to glucose intolerance as a result of impaired capacity to increase insulinemia in response to hyperglycemia. Pancreatic islets from these mice showed no obvious morphostructural abnormalities or decreased insulin content. In contrast, these islets failed to secrete insulin in response to glucose or insulin but were competent in succinate-, ketoisocaproate-, 3-isobutyl-1-methylxanthine (IBMX-), KCl-, and tolbutamide-induced insulin secretion. At the molecular level, JNX activation in beta-cells inhibited insulin-induced Akt phosphorylation, pancreatic and duodenal homeobox 1 nucleocytoplasmic shuttling, and transcription of insulin-target genes. Remarkably, rosiglitazone restored insulin secretion in response to hyperglycemia in mice and insulin-induced insulin secretion and signaling in isolated islets. In conclusion, the mere activation of JNK suffices to induce insulin resistance in pancreatic beta-cells by inhibition of insulin signaling in these cells, but it is not sufficient to elicit beta-cell death. In addition, we provide the first evidence that thiazolidinediones exert insulin-sensitizing action directly on pancreatic beta-cells.