AbstractGiven the ability of human serum albumin (HSA) to bind hydrophobic ligands, the binding mode of α‐tocopherol, the most representative member of the vitamin E family, is reported. α‐Tocopherol binds to HSA withK$_{\rm d}^0$= (7.0 ± 3.0) × 10−6M (pH 7.2, 25.0°C). Competitive and allosteric modulation of α‐tocopherol binding to full‐length and truncated (Asp1‐Glu382) HSA by endogenous and exogenous ligands suggests that it accommodates preferentially in the FA3–FA4 site. As HSA is taken up into cells, colocalizes with the α‐tocopherol transfer protein, and contributes to ligand secretion via ABCA1, it might participate in the distribution of α‐tocopherol between plasma, cells, and tissues. © 2013 BioFactors, 2013