The electrospinning of gelatin aqueous solution was successfully carried out by elevating the spinning temperature. The effects of spinning temperature and solution concentration were investigated on the morphology of gelatin nanofibers in the current study. To improve the stability and mechanical properties in moist state, the gelatin nanofibrous membrane was chemically crosslinked by 1-ethyl-3-dimethyl-aminopropyl carbodiimide hydrochloride and N-hydroxyl succinimide. The concentration of crosslinker was optimized by measuring the swelling degree and weight loss. Nanofibrous structure of the membrane was retained after lyophilization, although the fibers were curled and conglutinated. Tensile test revealed that the hydrated membrane becomes pliable and provides predetermined mechanical properties. Periodontal ligament cells cultured on the membrane in vitro exhibited good cell attachment, growth, and proliferation. Gelatin nanofibrous membrane can be one of promising biomaterials for the regeneration of damaged periodontal tissues.