The aim of this study is to enhance 3-hydroxyhexanoate (3HHx) fractions of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), abbreviated as PHBHHx, through site-directed mutagenesis of Aeromonas hydrophila enoyl Coenzyme A hydratase (PhaJ(Ah)). Two amino acids (Leu-65 and Val-130) were selected as a substitutional site based on the structural information of PhaJ(Ah). The purified proteins from the wild-type enzyme and mutants were used to determine hydratase activities. Hydratase activities of four single-mutation enzymes were similar to those of the wild type PhaJ(Ah), while hydratase activities of two double-mutation enzymes were much lower. In addition, the mutated phaJ (Ah) was individually co-transformed into E. coli BL21 (DE3) together with pFH21, which carried the PHA synthase (PhaC(Ah)) gene from A. hydrophila. The recombinant E. coli harboring plasmid pETJ1 (L65A), pETJ2 (L65V) or plasmid pETJ3 (V130A) synthesized the enhanced 3HHx fractions of PHBHHx from dodecanoate, indicating that Leu-65 and Val-130 of PhaJ(Ah) play an important role in determining the acyl chain length substrate specificity. The mutated PhaJ(Ah) (L65A, L65V, or V130A) provided higher 3HHx precursors for PHA synthase, resulting in the enhanced 3HHx fractions of PHBHHx. It is possible to change the acyl chain length substrate specificity of PhaJ through site-directed mutagenesis and produce PHBHHx with a wider range of alterable monomer composition.