Crossover of nitrogen from cathode to anode is inevitable in typical membranes used in PEM fuel cells. This crossovered nitrogen normally accumulates in the hydrogen recirculation system at anode side channels. Excessive buildup of nitrogen in the anode side lowers the relative hydrogen concentration and finally affects the performance of fuel cell stack. So it is very important to analysis the nitrogen gas crossover at various operating conditions. In this study, characterization of nitrogen gas crossover in PEM fuel cell stack was investigated. The mass spectroscopy (MS) has been applied to measure the amount of the crossovered nitrogen gas at the anode exit. Results show that nitrogen gas crossover rate was affected by current density, anode and cathode stoichiometric ratio and operating pressure. Current density, anode stoichiometric ratio and anode operating pressure do not affect nitrogen crossover rate but anode exit concentration of nitrogen. Cathode pressure and stoichiometric ratio largely affect the nitrogen crossover rate.