Sirtuin 3 (SIRT3) is a protein deacetylase regulating beta-cell function through inhibiting oxidative stress in obese and diabetic mice, but the detailed mechanism and potential effect of beta-cell-specific SIRT3 on metabolic homeostasis, and its potential effect on other metabolic organs, are unknown. We found that glucose tolerance and glucose-stimulated insulin secretion were impaired in high-fat diet (HFD)-fed beta-cell-selective Sirt3 knockout (Sirt3(f/f;Cre/+)) mice. In addition, Sirt3(f/f;Cre/+) mice had more severe hepatic steatosis than Sirt3(f/f) mice upon HFD feeding. RNA sequencing of islets suggested that Sirt3 deficiency overactivated 5-hydroxytryptamine (5-HT) synthesis as evidenced by upregulation of tryptophan hydroxylase 1 (TPH1). 5-HT concentration was increased in both islets and serum of Sirt3(f/f;Cre/+) mice. 5-HT also facilitated the effect of palmitate to increase lipid deposition. Treatment with TPH1 inhibitor ameliorated hepatic steatosis and reduced weight gain in HFD-fed Sirt3(f/f;Cre/+) mice. These data suggested that under HFD feeding, SIRT3 deficiency in beta-cells not only regulates insulin secretion but also modulates hepatic lipid metabolism via the release of 5-HT.