Immune cells contain high concentrations of vitamin C (ascorbate), which indicates an important role in immune function. In the intracellular environment, an important function of ascorbate is its capacity to act as a cofactor for iron (Fe)- and copper (Cu)-containing enzymes, many of which are now known to play a regulatory role in cell signaling processes, and in determining cellular differentiation and survival pathways. These enzymes include the proline and asparagine hydroxylases that regulate the constitutively expressed hypoxia-inducible factors (HIFs), which have been shown to modulate immune function at hypoxic inflammatory sites and in tumors. A number of recent studies have shown that ascorbate is able to modulate the hypoxic response, and its ability to influence immune function at inflammatory sites and in tumors may determine health outcomes. In addition, a large group of epigenetic demethylases with dependency on ascorbate for function has been identified. These enzymes include DNA and histone demethylases that have been found to be involved in the regulation of immune cell differentiation and phenotypic changes. The capacity for intracellular ascorbate to influence these immune cell functions is a focus of current research and will have important implications for our understanding of immune function and cancer.