BLX-1002 is a novel small thiazolidinedione with no apparent affinity to peroxisome proliferator-activated receptors (PPAR) that has been shown to reduce glycemia in type 2 diabetes without adipogenic effects. Its precise mechanisms of action, however, remain elusive and no studies have been done with respect to possible effects of BLX-1002 on pancreatic -cells. We have investigated the influence of the drug on -cell function in mouse islets in vitro. BLX-1002 enhanced insulin secretion stimulated by high, but not low or intermediate, glucose concentrations. BLX-1002 also augmented [Ca²⁺]_i at high glucose, an effect that was abolished by pre-treatment with the Ca²⁺-ATPase inhibitor thapsigargin. In contrast, BLX-1002 did not interfere with voltage-gated Ca²⁺ channel or with KATP channel activities. In addition, cellular NAD(P)H stimulated by glucose was not affected by the drug. The stimulatory effect of BLX-1002 on insulin secretion at high glucose was completely abolished by treatment with the phosphatidylinositol (PI) 3-kinase (PI3-K) inhibitors wortmannin or LY294002. Stimulation of the -cells with BLX-1002 also induced activation of AMPK at high glucose. Our study suggests that BLX-1002 potentiates insulin secretion only at high glucose in -cells in a PI3-K-dependent manner. This effect of BLX-1002 is associated with an increased [Ca²⁺]_i mediated through Ca²⁺ mobilization, and an enhanced activation of AMPK. The glucose-sensitive stimulatory impact of BLX-1002 on -cell function may translate into substantial clinical benefits of the drug in the management of type 2 diabetes, by avoidance of hypoglycemia.