tRNA-derived fragments (tRFs) are an emerging class of small non-coding RNAs with distinct cellular functions. Here, we studied the contribution of tRFs to the regulation of postnatal β cell maturation, a critical process that may lead to diabetes susceptibility in adulthood. We identified three tRFs abundant in neonatal rat islets originating from 5′ halves (tiRNA-5s) of histidine and glutamate tRNAs. Their inhibition in these islets reduced β cell proliferation and insulin secretion. Mitochondrial respiration was also perturbed, fitting with the mitochondrial enrichment of nuclear-encoded tiRNA-5^HisGTG and tiRNA-5^GluCTC. Notably, tiRNA-5 inhibition reduced Mpc1, a mitochondrial pyruvate carrier whose knock down largely phenocopied tiRNA-5 inhibition. tiRNA-5^HisGTG interactome revealed binding to Musashi-1, which was essential for the mitochondrial enrichment of tiRNA-5^HisGTG. Finally, tiRNA-5s were dysregulated in the islets of diabetic and diabetes-prone animals. Altogether, tiRNA-5s represent a class of regulators of β cell maturation, and their deregulation in neonatal islets may lead to diabetes susceptibility in adulthood.