Phasin protein (PhaP) is known to anchor into the matrix of phospholipid surrounding polyhydroxyalkanoic acid (PHA) inclusion bodies formed in bacterial cells and regulate the size of the granules, as well as the number of PHA granules. To investigate the effect of phasin on the formation of artificial poly(3-hydroxybutyrate) (P(3HB)) granules in vitro, (His)6-tagged or GST-fusion recombinant phasin was prepared and utilized for the artificial granule preparation. In addition, a P(3HB) depolymerase was coloaded with the recombinant phasin to prepare self-degradable phasin-coated P(3HB) granules. A water/chloroform two-phase emulsion technique was used, in which the emulsification was carried out by sonication, and the chloroform in the emulsion was removed by stirring-aided evaporation at room temperature or 65°C. Slower chloroform removal at room temperature produced better spherically shaped P(3HB) nanogranules, which were uniformly sized (∼100 to 200 nm in diameter). The self-degradability of P(3HB) depolymerase-loaded P(3HB) nanogranules was investigated. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 41074.