Polyhydroxyalkanoates (PHA) are polyesters of various hydroxyalkanoates accumulated in numerous bacteria. All of the monomeric units of PHA are enantiomerically pure and in R-configuration. R-Hydroxyalkanoic acids can be widely used as chiral starting materials in fine chemical, pharmaceutical and medical industries. In this study, we established an efficient method for the production of chiral hydroxyalkanoic acid monomers from PHA. Pseudomonas putida cells containing PHA were resuspended in phosphate buffer at different pH. We observed that the optimal initial pH for intracellular PHA degradation and monomer release was at pH 8-11 with pH 11 as the best. At initial pH 11, PHA containing 3-hydroxyoctanoic acid and 3-hydroxyhexanoic acid was degraded with an efficiency of over 90% (w/w) in 9 h, and the yield of the corresponding monomers was also over 90%. Under the same conditions, unsaturated monomers were also effectively produced from PHA containing 3-hydroxy-6-heptenoic acid, 3-hydroxy-8-nonenoic acid, and 3-hydroxy-10-undecenoic acid. The monomers (e.g., 3-hydroxyoctanoic acid) were further isolated using solid phase extraction and purified on reversed phase semipreparative liquid chromatography. We confirmed that the purified 3-hydroxyoctanoic acid monomer has exclusively the R-configuration.