After purification from the crude commercial preparation, the 3D structure of the synthetically valuable lipase from Pseudomonas stutzeri (LipC) is described through homology modelling, leading to a rational explanation of its catalytic behaviour. This elucidates that the enzyme has an active site defined by residues Ser-109, His-277 and Asp-255, and an oxyanion hole formed by peptidic NH groups from Met-43 and His-110. Interestingly, the active site is covered by two lids, one of them (Lid1, residues 145–181) being larger than the other (Lid2, residues 233–253). The opening and closing of these lids have been simulated by molecular modelling assuming both water and pure THF as solvents. Accordingly, THF clearly helps the exposure of the catalytic serine to the reaction medium which explains its excellent reported performance in this organic solvent. On the other hand, the stereospecificity of this enzyme is explained considering a small hydrophobic cavity formed by Gly-45, Phe-46, Tyr-54, Trp-55, Leu-278, Val-281 and Phe-284; particularly, Tyr-54 plays an important role in substrate recognition. In fact, in benzoin acylation, this residue forces the benzoyl group of the substrate to go into that cavity via H bonding with the carbonyl O atom of benzoin, thereby explaining the observed S-preference in benzoin acylation, which apparently contradicts the canonical Kazlauskas’ rule. For other alcohols non possessing the -hydroxycarbonyl core, Tyr-54 is allowing the entrance into the above-mentioned hydrophobic cavity only to those substrates with no steric hindrance in the vicinity of the hydroxymethane moiety.