The zinc air fuel cell (ZAFC) is a promising candidate for electrical energy storage and electric vehicle propulsion. However, its limited durability has become a major obstacle for its successful commercialization. In this study, 2-cell stacks, 25 cm² cells and three-electrode half-cells are constructed to experimentally investigate the degradation characteristics of the air cathode. The results of electrochemical tests reveal that the peak power density for the 25 cm2 cell with a new air cathode is 454 mW cm−2, which is twice as the value of the used air cathode. The electrochemical impedance analysis shows that both the charge transfer resistance and the mass transfer resistance of the used air cathodes have increased, suggesting that the catalyst surface area and gas diffusion coefficient have decreased significantly. Additionally, the microstructure and morphology of the catalytic layer (CL) and gas diffusion layer (GDL) are characterized by scanning electron microscopes (SEM). SEM results confirm that the micropores in CL and GDL of the used air cathode are seriously clogged, and many catalyst particles are lost. Therefore, the performance degradation is mainly due to the clogging of micropores and loss of catalyst particles. Furthermore, hypotheses of degradation mechanism and mitigation strategies for GDL and CL are discussed briefly.