Objective. As glucokinase (GK) is a metabolic sensor involved in the regulated release of insulin, we have investigated the acute actions of a novel GK activator – GKA50, on islet function. Research Design and Methods. Insulin secretion was determined by ELISA and microfluorimetry with fura-2 was used to examine intracellular Ca²⁺ homeostasis ([Ca²⁺]_i) in isolated mouse, rat and human islets of Langerhans, and in the MIN6 insulin-secreting cell line. Results. In rodent islets and MIN6 cells, 1μM GKA50 was found to stimulate insulin secretion and raise [Ca²⁺]_i in the presence of glucose (2-10mM). Similar effects on insulin release were also seen in isolated human islets (n=2 donors). GKA50 (1μM) caused a leftwards shift in the glucose-concentration response profiles and the EC₅₀ values for glucose were shifted by 3mM in rat islets (n=5) and approximately 10mM in MIN6 cells (n=7). There was no significant effect of GKA50 on the maximal rates of GSIS (n=7). In the absence of glucose GKA50 failed to elevate [Ca²⁺]_i (1μM GKA50) (n=4) or stimulate insulin release (30nM-10μM GKA50, n=3). At 5mM glucose the EC₅₀ for GKA50 in MIN6 cells was approximately 0.3μM (n=7). Inhibition of GK with mannoheptulose or 5-thioglucose selectively inhibited the action of GKA50 on insulin release, but not the effects of tolbutamide (n=3). Similarly, 3-methoxyglucose prevented GKA50-induced rises in [Ca²⁺]_i but not the actions of tolbutamide (n=10). Finally, the K_ATP channel agonist diazoxide (200μM) inhibited GKA50-induced insulin release (n=4) and its elevation of [Ca²⁺]_i (n=8). Conclusions. We show that GKA50 is a glucose-like activator of insulin-secreting cell metabolism in rodent and human islets and a Ca²⁺-dependent modulator of insulin secretion.