Increased glucose consumption is a hallmark of cancer cells. The increased consumption and subsequent metabolism of glucose during proliferation creates the need for a constant supply of NAD, a co-factor in glycolysis. Regeneration of the NAD required to support enhanced glycolysis has been attributed to the terminal glycolytic enzyme, lactate dehydrogenase (LDH). However, loss of glucose carbons to biosynthetic pathways early in glycolysis reduces the carbon supply to LDH. Thus, alternative routes for NAD regeneration must exist to support the increased glycolytic rate while allowing for the diversion of glucose to generate biomass and support proliferation. Here we demonstrate, using a variety of cancer cell lines as well as activated primary T cells, that cytosolic malate dehydrogenase 1 (MDH1) is an alternative to LDH as a supplier of NAD. Moreover, our results indicate that MDH1 generates malate with carbons derived from glutamine, thus enabling utilization of glucose carbons for glycolysis and for biomass. Amplification of MDH1 occurs at an impressive frequency in human tumors and correlates with poor prognosis. Together, our findings suggest that proliferating cells rely on both MDH1 and LDH to replenish cytosolic NAD, and that therapies designed at targeting glycolysis must consider both dehydrogenases.