Suboptimal intake of dietary vitamin C (ascorbate) increases the risk of several chronic diseases but the exact metabolic pathways affected are still unknown. In this study, we examined the metabolic profile of mice lacking the enzyme gulonolactone oxidase (Gulo) required for the biosynthesis of ascorbate. Gulo−/− mice were supplemented with 0%, 0.01%, and 0.4% ascorbate (w/v) in drinking water and serum was collected for metabolite measurements by targeted mass spectrometry. We also quantified 42 serum cytokines and examined the levels of different stress markers in liver. The metabolic profiles of Gulo−/− mice treated with ascorbate were different from untreated Gulo−/− and normal wild type mice. The cytokine profiles of Gulo−/− mice, in return, overlapped the profile of wild type animals upon 0.01% or 0.4% vitamin C supplementation. The life span of Gulo−/− mice increased with the amount of ascorbate in drinking water. It also correlated significantly with the ratios of serum arginine/lysine, tyrosine/phenylalanine, and the ratio of specific species of saturated/unsaturated phosphatidylcholines. Finally, levels of hepatic phosphorylated endoplasmic reticulum associated stress markers IRE1α and eIF2α correlated inversely with serum ascorbate and life span suggesting that vitamin C modulates endoplasmic reticulum stress response and longevity in Gulo−/− mice.