Pancreatic beta-cell response to glucose stimulation is governed by tightly regulated signaling pathways which have not been fully characterized. A screen for novel signaling intermediates identified Pim3 as a glucose-responsive gene in the beta-cell, and here, we characterize its role in the regulation of beta-cell function. Pim3 expression in the beta-cell was first observed through microarray analysis on glucose-stimulated murine insulinoma (MIN6) cells where expression was strongly and transiently induced. In the pancreas, Pim3 expression exhibited similar dynamics and was restricted to the beta-cell. Perturbation of Pim3 function resulted in enhanced glucose-stimulated insulin secretion, both in MIN6 cells and in isolated islets from Pim3(-/-) mice, where the augmentation was specifically seen in the second phase of secretion. Consequently, Pim3(-/-) mice displayed an increased glucose tolerance in vivo. Interestingly, Pim3(-/-) mice also exhibited increased insulin sensitivity. Glucose stimulation of isolated Pim3(-/-) islets resulted in increased phosphorylation of ERK1/2, a kinase involved in regulating beta-cell response to glucose. Pim3 was also found to physically interact with SOCS6 and SOCS6 levels were strongly reduced in Pim3(-/-) islets. Overexpression of SOCS6 inhibited glucose-induced ERK1/2 activation, strongly suggesting that Pim3 regulates ERK1/2 activity through SOCS6. These data reveal that Pim3 is a novel glucose-responsive gene in the beta-cell that negatively regulates insulin secretion by inhibiting the activation of ERK1/2, and through its effect on insulin sensitivity, has potentially a more global function in glucose homeostasis.