The adsorption of water and substrate on immobilized Candida antarctica lipase B was studied by performing adsorption isotherm measurements and using inverse gas chromatography (IGC). Water adsorption isotherm of the immobilized enzyme showed singular profile absorption incompatible with the Brunauer-Emmet-Teller model, probably due to the hydrophobic nature of the support, leading to very low interactions with water. IGC allowed determining the evolution with water thermodynamic activity (aW) of both dispersive surface energies and acidity and basicity constants of immobilized enzyme. These results showed that water molecules progressively covered immobilized enzyme, when increasing aW, leading to a saturation of polar groups above aW 0.1 and full coverage of the surface above aW 0.25. IGC also enabled relevant experiments to investigate the behavior of substrates under aW that they will experience, in a competitive situation with water. Results indicated that substrates had to displace water molecules in order to adsorb on the enzyme from aW values ranging from 0.1 to 0.2, depending on the substrate. As the conditions used for these adsorption studies resemble the ones of the continuous enzymatic solid/gas reactor, in which activity and selectivity of the lipase were extensively studied, it was possible to link adsorption results with particular effects of water on enzyme properties.