The influence of the wettability of the anode microporous layer (MPL) on both cell performance and operation duration of air-breathing direct methanol fuel cells (DMFCs) was investigated. The experimental results demonstrated that a passive DMFC with a hydrophilic MPL (DMFC-L) in the anode gas diffusion layer (GDL) showed performance superior to that with a hydrophobic MPL (DMFC-B), when the performance evaluation was conducted by applying a holding time of 45 s at each current density. DMFC-B showed good performance at medium and high current densities when a holding time of 150 s was used. The observation of water accumulation on the cathode of DMFC-L indicated that the decreased performance resulted mainly from blockage of the oxygen supply path. The constant-current discharging test showed that DMFC-B exhibited a lower performance at the beginning of discharge. However, it showed a lower rate of water accumulation on the cathode and thereby relatively stable operation. Operating the passive DMFCs at high water evaporation rate confirmed the important role of the wettability of anode GDLs for cathode oxygen transport.