Insulin is secreted from the islets of Langerhans in coordinated pulses. These pulses are thought to lead to plasma insulin oscillations, which are putatively more effective in lowering blood glucose than continuous levels of insulin. Gap-junction coupling of β-cells by connexin-36 coordinates intracellular free calcium oscillations and pulsatile insulin release in isolated islets, however a role in vivo has not been shown. We test whether loss of gap-junction coupling disrupts plasma insulin oscillations and whether this impacts glucose tolerance. We characterized the connexin-36 knockout (Cx36 -/-) mouse phenotype and performed hyperglycemic clamps with rapid sampling of insulin in Cx36 -/- and control mice. Our results show that Cx36 -/- mice are glucose intolerant, despite normal plasma insulin levels and insulin sensitivity. However, Cx36 -/- mice exhibit reduced insulin pulse amplitudes and a reduction in first-phase insulin secretion. These changes are similarly found in isolated Cx36 -/- islets. We conclude that Cx36 gap junctions regulate the in vivo dynamics of insulin secretion, which in turn is important for glucose homeostasis. Coordinated pulsatility of individual islets enhances the first-phase elevation and second-phase pulses of insulin. Because these dynamics are disrupted in the early stages of type 2 diabetes, dysregulation of gap-junction coupling could be an important factor in the development of this disease.