Microenvironments in which cells live play an important role in the attachment, growth and interactions of cells. To mimic the natural structure of extracellular matrices, electrospinning was applied to fabricate biomaterials into ultrafine fibers. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biocompatible and biodegradable polyester, has been shown to be an excellent biomaterial candidate for tissue engineering. In this study, five types of PHBV fibrous scaffolds with different diameters and orientations were obtained by changing solvents, concentration of electrospun solution and collector. Three kinds of scaffolds with good continuity and suitable mechanical properties, selected according to the morphology and mechanical properties of the scaffolds, were used for studying the influence of fiber diameter and orientation on growth behavior of bone-marrow-derived mesenchymal stem cells (MSCs). The results indicated that the random-oriented nanofibrous scaffold is most favorable for cell growth compared to other scaffolds, while the microfibrous scaffold resulted in the lowest viability of MSCs. The orientation of nanofibers showed a distinct effect on cell morphology by guiding cell skeleton extension. Both the random-oriented and aligned PHBV nanofibrous scaffolds showed to be good candidates for applications in tissue engineering.