Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and PHBV-gelatin were electrospun to obtain defect-free nanofibers by optimizing various process and solution parameters. Tensile strength, Young's modulus, and wettability of PHBV-gelatin nanofibrous scaffold were determined and compared with PHBV nanofibrous scaffold. Our results demonstrate that PHBV-gelatin nanofibers exhibited higher tensile strength and Young's modulus than the PHBV nanofibers. Human esophageal epithelial cells (HEEpiC) were cultured on PHBV and PHBV-gelatin nanofiber showed better cell proliferation in PHBV nanofibrous scaffold than the PHBV-gelatin scaffold after 7âdays of culture. HEEpiC cultured on PHBV and PHBV-gelatin nanofibrous scaffold exhibited characteristic epithelial cobblestone morphology after 3 days of culture. Further, the HEEpiC extracellular matrix (ECM) proteins (collagen type IV and laminin) and phenotypic marker proteins (cytokeratin-4 and 14) expressions were significantly higher in PHBV-gelatin nanofibrous scaffold than the PHBV nanofiber scaffold. However, the long-term stability and functional state of the cells on the PHBV scaffold give it an edge over the blend scaffolds. Thus, PHBV-based nanofibrous scaffolds could be explored further as ECM substitutes for the regeneration of esophageal tissue.