Viral infections can cause cellular dysregulation of metabolic reactions. Viruses alter host metabolism to meet their replication needs. The impact of viruses on specific metabolic pathways is not well understood, even for a well-studied virus-like human adenovirus. Adenoviral infection is known to affect cellular glycolysis and respiration, however, global effects on cellular metabolic pathways in response to adenoviral infection are lacking, particularly in normally quiescent structural cells, such as fibroblasts. Further, few studies have employed an untargeted approach with an emphasis on viral dosage and duration of infection. To address this, we employed untargeted metabolomics to quantify the dynamic metabolic shifts in fibroblasts infected with human adenovirus serotype 5 (HAdV-5) at three dosages (0.5, 1.0, and 2.0 multiplicity of infection [MOI]) and across four time points (6, 12, 24, and 36 h post-infection [HPI]). The greatest differences in individual metabolites were observed at 6– and 12-hours post-infection. In addition to its effects on glycolysis and respiration, adenoviral infection downregulated cysteine and unsaturated fatty acid metabolism, while upregulated purine metabolism. These results reveal the specific metabolic pathways that are perturbed by adenoviral infection and the associated dynamic shifts in metabolism, suggesting that viral infections alter energetics via profound changes in protein, lipid, and nucleic acid metabolism. The results revealed previously unconsidered metabolic pathways disrupted by HAdV-5 that can alter cells, even in non-excitable structural cells, such as fibroblasts.ImportanceHuman adenoviruses overtake the DNA replication machinery of the infected host, rewiring mitotic events and leading to effects on cellular respiration and glycolysis. Fibroblast lineages are normally quiescent cells that display a repertoire of responses to certain agonists. While metabolism often begins with glucose breakdown in the form of aerobic glycolysis, additional pathways are important for the overall functioning of the cell. Data on shifts in the metabolism of fibroblast cells in response to human adenoviral infection are lacking. We used an untargeted metabolomic approach to better understand the dynamic metabolic changes in human kidney cells in response to three viral dosages across four time points post infection. Profound shifts were observed for the cysteine, purine, and unsaturated fatty acid metabolites. This analysis provides a global perspective and highlights previously underappreciated aspects of how human adenoviruses alter host metabolism.