Sustainable production of bioplastics by heterotrophic microbes has been restricted by the limited resources of organic substrates and the energy required for biomass harvest. Here, the easy-to-harvest cyanobacterium (Chlorogloea fritschii TISTR 8527), from which the biomass instantaneously settled to the bottom of liquid culture, was utilized to produce poly-3-hydroxybutyrate (PHB) using a two-stage cultivation strategy. The cells were first pre-grown under normal photoautotrophy to increase their biomass and then recultivated under a heterotrophic condition with a single organic substrate to produce the product. Through optimization of this two-stage cultivation, the mass conversion efficiency of acetate substrate to PHB was obtained at 51 ± 7% (w/w), the comparable level to the theoretical biochemical conversion efficiency of acetate to PHB. This two-stage cultivation that efficiently converted the substrate to the product, concurrent with a reduced culture biomass, may be applicable for the production of other biopolymers by cyanobacteria.