The anode gas diffusion layer plays an important role on mass transport. By adjusting the assembly pressure, the structure of gas diffusion layer can be controlled to investigate the two-phase behavior. The electrochemical test indicates that increasing the assembly pressure causes increase in mass transport resistance. Inhomogeneous compression of gas diffusion layer is observed by three-dimensional imaging technology. Based on the experimental data, a two-phase model is established. The simulated results show that the increment of surface roughness and the reduction of porosity, both derived from compression of gas diffusion layer, exacerbate CO2 blockage of the cell. Lowering polytetrafluoroethylene content of anode gas diffusion layer alleviates CO2 blockage by reducing the difference of surface tension between liquid-solid and gas-solid interface as well as the negative capillary pressure of gas diffusion layer. Thus, the peak power density climbs from 60.61 to 49.94 mW cm−2 to 74.79 and 70.47 mW cm−2 at the assembly pressure of 1.00 and 2.00 MPa, respectively. The optimal assembly pressure locates at where good contact between membrane electrode assembly and bipolar plate is achieved, with the least gas diffusion layer deformation possible.