Vitamin C (ascorbate) is an essential water-soluble micronutrient in humans and is obtained through the diet, primarily from fruits and vegetables. In vivo, vitamin C acts as a cofactor for numerous biosynthetic enzymes required for the synthesis of amino acid-derived macromolecules, neurotransmitters, and neuropeptide hormones, and is also a cofactor for various hydroxylases involved in the regulation of gene transcription and epigenetics. Vitamin C was first chemically synthesized in the early 1930s and since then researchers have been investigating the comparative bioavailability of synthetic versus natural, food-derived vitamin C. Although synthetic and food-derived vitamin C is chemically identical, fruit and vegetables are rich in numerous nutrients and phytochemicals which may influence its bioavailability. The physiological interactions of vitamin C with various bioflavonoids have been the most intensively studied to date. Here, we review animal and human studies, comprising both pharmacokinetic and steady-state designs, which have been carried out to investigate the comparative bioavailability of synthetic and food-derived vitamin C, or vitamin C in the presence of isolated bioflavonoids. Overall, a majority of animal studies have shown differences in the comparative bioavailability of synthetic versus natural vitamin C, although the results varied depending on the animal model, study design and body compartments measured. In contrast, all steady state comparative bioavailability studies in humans have shown no differences between synthetic and natural vitamin C, regardless of the subject population, study design or intervention used. Some pharmacokinetic studies in humans have shown transient and small comparative differences between synthetic and natural vitamin C, although these differences are likely to have minimal physiological impact. Study design issues and future research directions are discussed.