Polyhydroxyalkanoate depolymerase derived from Variovorax sp. C34 (PhaZ(Vs)) was identified as the first enzyme that is capable of degrading isotactic P[67 mol% (R)-lactate(LA)-co-(R)-3-hydroxybutyrate(3HB)] [P(d-LA-co-d-3HB)]. This study aimed at analyzing the monomer sequence specificity of PhaZ(Vs) for hydrolyzing P(LA-co-3HB) in comparison with a P(3HB) depolymerase from Alcaligenes faecalis T1 (PhaZ(Af)) that did not degrade the same copolymer. Degradation of P(LA-co-3HB) by action of PhaZ(Vs) generated dimers, 3HB-3HB, 3HB-LA, LA-3HB, and LA-LA, and the monomers, suggesting that PhaZ(Vs) cleaved the linkages between LA and 3HB units and between LA units. To provide a direct evidence for the hydrolysis of these sequences, the synthetic methyl trimers, 3HB-3HB-3HB, LA-LA-3HB, LA-3HB-LA, and 3HB-LA-LA, were treated with the PhaZs. Unexpectedly, not only PhaZ(Vs) but also PhaZ(Af) hydrolyzed all of these substrates, namely PhaZ(Af) also cleaved LA-LA linkage. Considering the fact that both PhaZs did not degrade P[(R)-LA] (PDLA) homopolymer, the cleavage capability of LA-LA linkage by PhaZs was supposed to depend on the length of the LA-clustering region in the polymer chain. To test this hypothesis, PDLA oligomers (6 to 40 mer) were subjected to the PhaZ assay, revealing that there was an inverse relationship between molecular weight of the substrates and their hydrolysis efficiency. Moreover, PhaZ(Vs) exhibited the degrading activity toward significantly longer PDLA oligomers compared to PhaZ(Af). Therefore, the cleaving capability of PhaZs used here toward the d-LA-based polymers containing the LA-clustering region was strongly associated with the substrate length, rather than the monomer sequence specificity of the enzyme.