Na-O2 batteries have drawn increasing attention in recent years, owing to their high energy density and the abundance of sodium resources. Their applications still suffer, however, from lack of effective air cathodes to achieve a stable and long cycle life. Herein, we report a nitrogen-doped carbon nanofiber (NCF) material derived from polypyrrole as air cathode for the non-aqueous Na-O2 electrochemical system. Notably, Na-O2 batteries with NCF as air cathode could achieve a low overpotential gap of 500 mV, a high specific capacity of 8554.7 mA h g−1 at 100 mA g−1, and excellent cyclic stability over 90 cycles with NaO2 as the discharge product. These excellent performances can be attributed to the combination of their highly conductive three-dimensional network structure, large surface area, and superior catalytic activity, obtained by incorporating nitrogen atoms into the carbon matrix, which can facilitate electron transportation, oxygen and electrolyte diffusion, and discharge product deposition and decomposition. Besides, density functional theory (DFT) calculations indicated that pyrrolic and pyridinic-N doping can effectively optimize the surface adsorption energy of the reactants and intermediate, which facilitate to achieve excellent oxygen reduction reaction (ORR) and oxygen evolution reaction (OER) activity. The results reported here can point the way to the rational design of electrocatalytic air cathodes for rechargeable Na-O2 batteries.