The binding preference of a polyhydroxyalkanoate (PHA) biosynthesis-related multifunctional protein from Ralstonia eutropha (PhaM(Re)) was characterized. In vitro activity assay showed that PHA synthase from R. eutropha (PhaC(Re)) was activated by the presence of PhaM(Re) but PHA synthase from Aeromonas caviae (PhaC(Ac)) was not. Additionally, in vitro assays of protein-protein interactions demonstrated that PhaM(Re) interacted with PhaC(Re) directly, but did not interact with PhaC(Ac). These results suggest that the protein-protein interaction is important for the activation of PhaC by PhaM(Re). Further analyses indicated that PhaM(Re) has little or no direct interaction with the PHA polymer chain. Subsequently, PHA biosynthesis genes (phaA (Re), phaB (Re), and phaC (Re)/phaC (Ac)) and the phaM (Re) gene were introduced into recombinant Escherichia coli and cultivated for PHA accumulation. Contrary to our expectations, the expression of PhaM(Re) decreased PHA accumulation and changed the morphology of PHA granules to be microscopically obscure shape in PhaC(Re)-expressing E. coli. No change in the amount of P(3HB) or the morphology of granules by PhaM(Re) expression was observed in PhaC(Ac)-expressing E. coli. These observations suggest that PhaM(Re) affects cellular physiology through the PhaM-PhaC interaction.