A recombinant Serratia sp. W3 lipase (rSmL) was expressed in E. coli and purified to homogeneity. The kinetic properties, regio-selectivity, and interfacial performances of rSmL were compared with those of the native lipase (nSmL). rSmL has a molecular mass of 67 kDa and a specific activity of 3530 U mg(-1) which is around 12-fold higher than that of nSmL (300 U mg(-1)) when using olive oil as the substrate. Both rSmL and nSmL were able to hydrolyze the ester bond at the sn-1 and sn-3 positions but exhibited a clear regio-preference towards the sn-3 position of the surface-coated triglycerides (TG), which were esterified with alpha-eleostearic acid at the sn-1/3 position or dicaprin isomers spread as monomolecular films. Molecular modeling and docking of TG into the active site of rSmL indicated that this regio-preference may be due to steric hindrance, created by the residues Ile(308) and Trp(311), with distances between the sn-1 reactive carbon and the catalytic Serine residue ranging from 3.15 to 5.47 angstrom. In contrast, the sn-3 positioning within the enzyme catalytic pocket resulted in shorter distances, ranging from 2.79 to 4.15 angstrom, therefore facilitating the hydrolysis at the sn-3 position.