The effect of 2'-differing substituent groups (such as the H, F, Cl, Br, OCH3) as well as various chain lengths of the acyl substrates (C6, C10, and C14) on the performance of Thermomyces lanuginosus lipase for the regioselective acylation of pyrimidine nucleoside analogs was investigated systematically for the first time. The results evidently demonstrated that changing substituents in nucleosides had a significant influence on the substrate specificity for the enzymatic reaction, possibly due to the differences in physicochemical properties of the substituents and the special shape of the substrate binding site in enzyme active region. With respect to the chain-length selectivity, the enzyme exhibited the highest 5'-regioselectivity toward the longer chain (C14) as compared to short (C6 and C10) ones. The reason might be attributed to the presence of the interaction between the acyl chain and the large hydrophobic substrate-binding pocket.