Diabetes can be caused by an insufficiency in β-cell mass. Here, we performed a genetic screen in a zebrafish model of β-cell loss to identify pathways promoting β-cell regeneration. We found that both folate receptor 1 (folr1) overexpression and treatment with folinic acid, stimulated β-cell differentiation in zebrafish. Treatment with folinic acid also stimulated β-cell differentiation in cultures of neonatal pig islets, showing that the effect could be translated to a mammalian system. In both zebrafish and neonatal pig islets, the increased β-cell differentiation originated from ductal cells. Mechanistically, comparative metabolomic analysis of zebrafish with/without β-cell ablation and with/without folinic acid treatment indicated β-cell regeneration could be attributed to changes in the pyrimidine, carnitine, and serine pathways. Overall, our results suggest evolutionarily conserved and previously unknown roles for folic acid and one-carbon metabolism in the generation of β-cells.