Thyroid hormone (TH) signaling promotes tissue maturation and adult organ formation. Developmental transitions alter an organism's metabolic requirements, and it remains unclear how development and metabolic demands are coordinated. We used the zebrafish as a model to test whether and how TH signaling affects pancreatic islet maturation, and consequently glucose homeostasis, during the larval to juvenile transition. We found that exogenous TH precociously activates the β-cell differentiation genes pax6b and mnx1 while downregulating arxa, a master regulator of α-cell development and function. Together, these effects induced hypoglycemia, at least in part by increasing insulin and decreasing glucagon expression. We visualized TH target tissues using a novel TH-responsive reporter line and found that both α- and β-cells become targets of endogenous TH signaling during the larval-to-juvenile transition. Importantly, endogenous TH is required during this transition for the functional maturation of α- and β-cells in order to maintain glucose homeostasis. Thus, our study sheds new light on the regulation of glucose metabolism during major developmental transitions.