To study effects of Bcl-x_L in the pancreatic β-cell, two transgenic lines were produced using different forms of the rat insulin promoter. Bcl-x_Lexpression in β-cells was increased 2- to 3-fold in founder (Fd) 1 and over 10-fold in Fd 2 compared with littermate controls. After exposure to thapsigargin (10 μM for 48 h), losses of cell viability in islets of Fd 1 and Fd 2 Bcl-x_Ltransgenic mice were significantly lower than in islets of wild-type mice. Unexpectedly, severe glucose intolerance was observed in Fd 2 but not Fd 1 Bcl-x_L mice. Pancreatic insulin content and islet morphology were not different from control in either transgenic line. However, Fd 2 Bcl-x_L islets had impaired insulin secretory and intracellular free Ca²⁺([Ca²⁺]_i) responses to glucose and KCl. Furthermore, insulin and [Ca²⁺]_iresponses to pyruvate methyl ester (PME) were similarly reduced as glucose in Fd 2 Bcl-x_L islets. Consistent with a mitochondrial defect, glucose oxidation, but not glycolysis, was significantly lower in Fd 2 Bcl-x_L islets than in wild-type islets. Glucose-, PME-, and α-ketoisocaproate-induced hyperpolarization of mitochondrial membrane potential, NAD(P)H, and ATP production were also significantly reduced in Fd 2 Bcl-x_L islets. Thus, although Bcl-x_L promotes β-cell survival, high levels of expression of Bcl-x_L result in reduced glucose-induced insulin secretion and hyperglycemia due to a defect in mitochondrial nutrient metabolism and signaling for insulin secretion.