CD36 is a multifunctional protein that enhances cellular fatty acid (FA) uptake, a key step in energy metabolism, and its dysregulation in multiple tissue sites is central to obesity-linked diabetes, a risk factor for atherosclerosis. Although CD36 has been implicated in FA uptake in a correlative way, the molecular mechanisms are not known. Their elucidation in cells is confounded by receptor-mediated uptake of low-density lipoprotein by CD36 and the competitive and/or contributive effects of other proteins involved in FA transport and metabolism, which include caveolin(s), fatty acid transport protein (FATP), intracellular fatty acid binding protein, and enzymes involved in the conversion of FAs to esters. Here we utilized a simpler cellular system (HEK cells), which lack caveolin-1, CD36, and FATP and metabolize FAs slowly compared to the time frame of transmembrane FA movement. Our previous studies of HEK cells showed that caveolin-1 affects FA binding and translocation across the plasma membrane and but not FA esterification [Simard, J. R., et al. (2010) J. Lipid Res. 51 (5), 914-922]. Our key new finding is that CD36 accelerates FA uptake and extensive incorporation into triglycerides, a process that is slower (minutes) than transmembrane movement (seconds). Real-time fluorescence measurements showed that the rates of binding and transport of oleic acid into cells with and without CD36 were not different. Thus, CD36 enhances intracellular metabolism, i.e., esterification, and thereby increases the rate of FA uptake without catalyzing the translocation of FA across the plasma membrane, suggesting that CD36 is central to FA uptake via its effects on intracellular metabolism.