The mechanism of enzymatic hydrolysis for (R)-3-hydroxybutyrate (3HB) oligomers with poly[(R)-3-hydroxybutyrate] [P(3HB)] depolymerase (PhaZpst) from Pseudomonas stutzeri was investigated by two deletion mutants lacking the substrate-binding domain and linker region, PhaZpst Delta sbd and PhaZpstcore. The two deletion mutants had no ability for hydrolysis of water-insoluble P(3HB), while the hydrolysis activities of two deletion mutants for water-soluble 3HB oligomer and its derivatives (dimer, trimer, and tetramer) were identical with those of the wild type, indicating that the function of catalytic domain is independent of its substrate-binding domain and linker region. The hydrolyzed products analysis of 3HB oligomers by HPLC showed that the active site of catalytic domain recognizes at least two 3HB units for hydrolysis. The initial rates of hydrolysis of dimer derivative were lower by 2 orders of magnitude than those of trimer and tetramer derivatives, suggesting that 3HB oligomer derivatives larger than trimer are favorite substrates for PhaZpst.