Humans utilize two sodium-ascorbate co-transporters (hSVCT1 & 2) for transporting the dietary essential micronutrient ascorbic acid, the reduced and active form of vitamin C. Although the human liver plays a pivotal role in regulating and maintaining vitamin C homeostasis, vitamin C transport physiology and regulation of the hSVCT systems in this organ have not been well defined. Thus, this report utilized a human hepatic cell line (HepG2), confirming certain results with primary human hepatocytes and determined the initial rate of ascorbic acid uptake to be Na⁺ gradient, pH dependent and saturable as a function of concentration over low and high micro-molar ranges. Additionally, hSVCT2 protein and mRNA are expressed at higher levels in HepG2 cells and native human liver and the cloned hSVCT2 promoter has more activity in HepG2 cells. Results using siRNA suggest that in HepG2 cells decreasing hSVCT2 message levels reduces the overall ascorbic acid uptake process more than decreasing hSVCT1 message levels. Activation of PKC intracellular regulatory pathways caused a down-regulation in ascorbic acid uptake not mediated by a single predicted PKC specific amino acid phosphorylation site in hSVCT1 or 2. However, PKC activation causes internalization of hSVCT1 but not hSVCT2. Examination of other intracellular regulatory pathways on ascorbic acid uptake determined that regulation also potentially occurs by PKA, PTK and Ca²⁺/CaM, but not by NO dependant pathways. These studies are the first determination of the overall ascorbic acid uptake process and relative expression, regulation and contribution of the hSVCT systems in human liver epithelial cells.