To avoid using toxic organic solvents and surfactants, production of chitosan-based nanofibers usually requires a concentrated acetic acid solution as solvent. In this study, chitosan-gelatin-polyvinyl alcohol (PVA) hybrid nanofibrous mats were successfully fabricated using only a mild concentration (20 wt%) of acetic acid by electrospinning. The optimum weight ratio of chitosan:gelatin:PVA was 2:2:4 which produced fiber diameters of around 150 nm. The chitosan-gelatin-PVA hybrid nanofibrous mats were further reinforced by crosslinking using glutaraldehyde vapor for 12 h. The crosslinked mats exhibited better stability and tensile strength (229.6 +/- 39.1 N/g). The DSC thermograms indicated that good homogeneity of the nanofibrous mats was achieved. The cytocompatibility test revealed better proliferation of mesenchymal stem cells on the nanofibrous mats than on the sponges. Due to enhanced strength, stability and cytocompatibility of the mats, and a mild acid solution required, the electrospun chitosan-gelatin-PVA hybrid nanofibrous mats show excellent potential for tissue engineering-related applications. (C) 2013 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.