Polyhydroxybutyrate (PHB) is a polymer used to restore tissues or regenerate bones. However, its compatibility with human bone marrow-derived mesenchymal stem cells (MSCs) has not been examined. PHB membranes of random (r-PHB) or aligned (a-PHB) electrospun nanofibers that generate bone and spinal axon scaffolds were combined with human MSCs. The adhesion and proliferation of cells on these membranes were examined. The orientation of cells on PHB membranes was analyzed by confocal microscopy and scanning electron microscopy (SEM). The MSCs maintained their characteristic properties on the membranes, adhered to the membranes, and preserved their viability. The cell morphology was different when they were grown on differentially designed matrices. Cells expanded on the a-PHB membrane and showed fibroid-like morphology. Conversely, cells interacting with the r-PHB membrane were located homogeneously and demonstrated polygonal morphology. The adhesion and proliferation of human MSCs was higher on the a-PHB membrane than on the randomly oriented one. Fiber orientation influenced the phenotype and biological behavior of human MSCs. This property may be useful in the selection of specifically designed scaffolds for the desired tasks. The results of this preliminary study indicated that PHB membranes designed for bone or nerve tissue engineering are compatible with human MSCs.