Chitosan nanofibers were electrospun from aqueous chitosan solution using concentrated acetic acid solution as a solvent. A uniform nanofibrous mat of average fiber diameter of 130 nm was obtained from the following optimum condition: 7% chitosan solution in aqueous 90% acetic acid solution was successfully electrospun in the electric field of 4 kV/cm. The aqueous acetic acid concentration higher than 30% was prerequisite for chitosan nanofiber formation, because more concentrated acetic acid in water progressively decreased surface tension of the chitosan solution and concomitantly increased charge density of jet without significant effect on solution viscosity. However, acetic acid solution more than 90% did not dissolve enough chitosan to make spinnable viscous concentration. Only chitosan of a molecular weight of 106,000 g/mol produced bead-free chitosan nanofibers, while low- or high-molecular-weight chitosans of 30,000 and 398,000 g/mol did not. Average fiber diameters and size distribution decreased with increasing electric field and more bead defects appeared at 5 kV/cm or more.