Systemic metabolic dysregulation in sepsis critically impacts patient survival. To better understand its onset, untargeted serum metabolomics and lipidomics are analyzed from 152 presymptomatic patients undergoing major elective surgery, and identified key metabolites, including serine and aminoadipic acid, that differentiate postoperative uncomplicated infection from sepsis. Using single-nucleus RNA sequencing data from an in vivo mouse model of sepsis, tissue-independent down-regulation and tissue-specific differences of serine and energy-related genes including key module roles for the mitochondria-linked genes, Cox4i1, Cox8a, and Ndufa4 are identified. Finally, serine-dependent metabolic shifts, especially in the liver, are revealed by using ¹²C/¹³C murine data with labeled serine, and link altered activity of the serine hydroxymethyltransferase (SHMT) cycle with perturbed purine metabolism during sepsis. This study demonstrates the close interrelationship between early metabolite changes and mitochondrial dysfunction in sepsis, improves the understanding of the underlying pathophysiology, and highlights metabolic targets to prospectively treat presymptomatic, but at-risk, patients.