Mitochondria constantly undergo fusion and fission events, referred as mitochondrial dynamics, which determine mitochondrial architecture and bioenergetics. Cultured cell studies demonstrate that mitochondrial dynamics are acutely regulated by phosphorylation of the mitochondrial fission orchestrator dynamin-related protein 1 (Drp1) at S579 or S600. However, the physiological impact and crosstalk of these phosphorylation sites is poorly understood. Here, we describe the functional interrelation between S579 and S600 phosphorylation sites in vivo and their role on mitochondrial remodeling. Mice carrying a homozygous Drp1 S600A knockin (Drp1 KI) mutation display larger mitochondria and enhanced lipid oxidation and respiratory capacities, granting improved glucose tolerance and thermogenic response upon high-fat feeding. Housing mice at thermoneutrality blunts these differences, suggesting a role for the brown adipose tissue in the protection of Drp1 KI mice against metabolic damage. Overall, we demonstrate crosstalk between Drp1 phosphorylation sites and provide evidence that their modulation could be used in the treatment and prevention of metabolic diseases. Display omitted •Drp1 phosphorylation at S600 promotes the phosphorylation at the S579 site•Both Drp1 P-S600 and P-S579 are required for maximal mitochondrial fragmentation•Drp1 S600A knockin mice are protected against diet-induced metabolic damage•Drp1 phosphorylation controls brown adipose tissue thermogenic capacity in mice Valera-Alberni et al. show that in mouse cells and tissues, Drp1 phosphorylation at S600 prompts the downstream phosphorylation of S579, triggering mitochondrial fission. Accordingly, Drp1 S600A knockin (Drp1 KI) mice display enlarged mitochondria. Metabolically, Drp1 KI mice show increased lipid oxidation capacity and are protected against diet-induced metabolic disease.