Cholesterol oxidase (ChOx) is a flavoenzyme that oxidizes and isomerizes cholesterol (CHL) to form cholest‐4‐en‐3‐one. Molecular docking and molecular dynamics simulations were conducted to predict the binding interactions of CHL in the active site. Several key interactions (E361‐CHL, N485‐FAD, and H447‐CHL) were identified and which are likely to determine the correct positioning of CHL relative to flavin‐adenine dinucleotide (FAD). Binding of CHL also induced changes in key residues of the active site leading to the closure of the oxygen channel. A group of residues, Y107, F444, and Y446, known as the hydrophobic triad, are believed to affect the binding of CHL in the active site. Computational site‐directed mutagenesis of these residues revealed that their mutation affects the conformations of key residues in the active site, leading to non‐optimal binding of CHL and to changes in the structure of the oxygen channel, all of which are likely to reduce the catalytic efficiency of ChOx. Proteins 2017; 85:1645–1655. © 2017 Wiley Periodicals, Inc.