Recent studies suggest that sphingolipid metabolism is altered during type 2 diabetes. Increased levels of the sphingolipid ceramide are associated with insulin resistance. However, a role for sphingolipids in pancreatic beta cell function, or insulin production, and release remains to be established. Our studies in MIN6 cells and mouse pancreatic islets demonstrate that glucose stimulates an intracellular rise in the sphingolipid, sphingosine 1-phosphate (S1P), whereas the levels of ceramide and sphingomyelin remain unchanged. The increase in S1P levels by glucose is due to activation of sphingosine kinase 2 (SphK2). Interestingly, rises in S1P correlate with increased glucose-stimulated insulin secretion (GSIS). Decreasing S1P levels by treatment of MIN6 cells or primary islets with the sphingosine kinase inhibitor reduces GSIS. Moreover, knockdown of SphK2 alone results in decreased GSIS, whereas knockdown of the S1P phosphatase, Sgpp1, leads to a rise in GSIS. Treatment of mice with the sphingosine kinase inhibitor impairs glucose disposal due to decreased plasma insulin levels. Altogether, our data suggest that glucose activates SphK2 in pancreatic beta cells leading to a rise in S1P levels, which is important for GSIS. Background: Sphingolipids play an important role in glucose homeostasis. Results: High glucose induces SphK activity, leading to increases in S1P levels and stimulation of insulin secretion. Conclusion: SphK activity and S1P levels are critical for glucose-stimulated insulin secretion. Significance: The presented data uncover a new role for SphK and S1P in regulation of insulin secretion by glucose.