The Escherichia coli fabGEc gene and the Pseudomonas aeruginosa rhlGPa gene, which encode 3-ketoacyl-acyl carrier protein reductase, were expressed in E. coli W3110 and its fadA mutant strain WA101 to examine their roles in medium-chain-length (MCL) polyhydroxyalkanoate (PHA) biosynthesis from fatty acids. When one of these 3-ketoacyl-acyl carrier protein reductase genes was co-expressed with the Pseudomonas sp. 61–3 PHA synthase gene (phaC2Ps) in E. coli W3110, MCL-PHA composed mainly of 3-hydroxyoctanoate and 3-hydroxydecanoate was synthesized from sodium decanoate. When the fabGEc gene and the phaC2Ps gene were co-expressed in the fadA mutant E. coli strain WA101, MCL-PHA rich in 3-hydroxydecanoate monomer up to 93 mol% was accumulated from sodium decanoate. This was possible by efficiently redirecting 3-ketoacyl-coenzymes A from the β-oxidation pathway to the PHA biosynthesis pathway without losing two carbon units, the strategy of which can be extended for the production of MCL-PHAs rich in other specific monomers.