Poly-3-hydroxybutyrate (PHB) is a biological polyester present in bacteria and eukaryotic cells. Long-chain (or storage) sPHB (up to 100,000 residues) is typically present in PHB-accumulating bacteria and localized in specialized granules known as carbonosomes. In these organisms, sPHB plays a major role as carbon and energy storage. On the other hand, short-chain (or complexed) cPHB (10–100 residues) is present in eukaryotic organisms, including mammals as well as in many bacteria. Previous studies indicated that cPHB is localized in various subcellular compartments of the eukaryotic organisms. Here, we used fluorescent microscopy to directly investigate the localization of PHB in mammalian cells. PHB was visualized in cultured U87 cells using fluorescent probe BODIPY 493/503. Specificity of PHB staining was confirmed by markedly decreased fluorescence of samples treated with PHB-specific depolymerase (PhaZ7). We found that PHB is associated with granules, and that these PHB-enriched granules do not co-localized with mitochondria, lysosomes, or endoplasmic reticulum. These results suggest that, in mammalian cells, PHB can accumulate in the cytoplasm in granules similar to ‘energy storage’ carbonosomes found in PHB-accumulating bacteria.