Some microbial lipases bio-imprinted with the reversible lipase inhibitor, Orlistat. Comparison of interesterification activities indicated that Orlistat bio-imprinting is equally effective as amphiphile imprinting and yields a 3.5–4.0-fold activity enhancement. Solvent-free incubation medium was as effective as hexane medium. Water addition into the incubation medium erased the bio-imprinting effect; hence, lid opening should be the mechanism of activation. This study provides additional proof to the hypothesis that lipase bio-imprinting is an active-site related phenomenon.