A biosynthetic pathway for the production of (R)-3-hydroxyalkanoates (R3HAs) through in vivo depolymerization of poly(3-hydroxyalkanoates) [P(3HAs)] was constructed in recombinant Escherichia coli fadA mutant WA101 by introducing the Pseudomonas sp. 61-3 PHA synthase gene (phaC2) and the P. aeruginosa intracellular PHA depolymerase gene (phaZ). When recombinant E. coli WA101 strain expressing the phaC2 gene and the phaZ gene was cultured in Luria-Bertani (LB) medium containing 2 g/L of sodium decanoate, R3HAs could be produced to the concentration of 0.49 g/L. The mole fraction of R3HAs was 7.5mol% of 3-hydroxybutyrate (3HB), 31.6mol% of 3-hydroxyhexanoate (3HHx), 30 mol% of 3-hydroxyoctanoate (3HO), 29.4mol% of 3-hydroxydecanoate (3HD), and 1.5mol% of 3-hydroxydodecanoate (3HDD). When the E. coli 3-ketoacyl-ACP reductase gene (fabG) was overexpressed to provide more (R)-3-hydroxyacyl-CoA (R3HA-CoA), the concentration of R3HAs was increased up to 1.05 g/L. Also, expression of the fabG gene resulted in the mole fraction change of produced R3HAs, in which 3HD fraction was enriched from 29.4mol% to 57.9mol% with the decrease of 3HHx fraction from 31.6mol% to 9.6mol%. Interestingly, the only expression of the fabG gene in E. coli WA101 could produce R3HAs to 0.55 g/L, which suggests that E. coli might have unidentified CoA hydrolases that have substrate specificities toward R3HA-CoA. This study shows the enantiomerically pure RHAs can be efficiently produced by metabolically engineered E. coli with high yield.