Under normal physiological conditions, glucagon is released from pancreatic alpha cells to elevate circulating glucose levels in response to hypoglycemia. In patients with type 2 diabetes, glucagon secretion is dysregulated, but the underlying mechanisms remain unclear. Several hypotheses have been suggested to explain the coupling of blood glucose sensing to electrical activity and glucagon secretion from alpha cells. Here, we show that glucose rapidly regulates mitochondrial motility and localization in alpha cells. Under conditions of low glucose, mitochondria are arrested in positions further from the nucleus, correlating with increased ATP/ADP in the sub-plasma membrane space. We also find that knockdown (KD) of Mitochondrial Rho GTPase 2 (Miro2), but not Miro1, reduces mitochondrial motility in alpha cells and impairs glucose-induced inhibition of glucagon secretion without effects on insulin secretion or mitochondrial motility in non-alpha islet cells. These findings highlight the significance of mitochondrial motility for alpha cell function and reveal fundamental differences between alpha and beta cells.