Naive CD8+ T cells differentiating into effector T cells increase glucose uptake and shift from quiescent to anabolic metabolism. Although much is known about the metabolism of cultured T cells, how T cells use nutrients during immune responses in vivo is less well defined. Here, we combined bioenergetic profiling and 13C-glucose infusion techniques to investigate the metabolism of CD8+ T cells responding to Listeria infection. In contrast to in vitro-activated T cells, which display hallmarks of Warburg metabolism, physiologically activated CD8+ T cells displayed greater rates of oxidative metabolism, higher bioenergetic capacity, differential use of pyruvate, and prominent flow of 13C-glucose carbon to anabolic pathways, including nucleotide and serine biosynthesis. Glucose-dependent serine biosynthesis mediated by the enzyme Phgdh was essential for CD8+ T cell expansion in vivo. Our data highlight fundamental differences in glucose use by pathogen-specific T cells in vivo, illustrating the impact of environment on T cell metabolic phenotypes. Display omitted •Developed 13C-infusion method for studying T cell metabolism in vivo•T cell glucose use and bioenergetics differ between cell culture and mouse models•Glucose metabolism in T cells changes dynamically over an immune response•Glucose-dependent serine biosynthesis supports T cell proliferation in vivo Although much is known about the metabolism of cultured T cells, how T cells use nutrients during immune responses in vivo is not well defined. Using 13C-glucose isotope tracing in mice, Ma et al. demonstrate prominent use of glucose for anabolic metabolism by T cells in vivo. This work highlights the importance of studying T cell metabolism in a physiological environment.