This study systematically investigates the effects of structural aspects on the performance of a passive air-breathing direct methanol fuel cell (DMFC). Three factors are selected in this study: (1) two different open ratios of the current collector; (2) two different assembly methods of the diffusion layer; and (3) three membrane types with different thicknesses. The interrelations and interactions among these factors have been taken into account. The results demonstrate that these structural factors combine to significantly affect the cell performance of DMFCs. The higher open ratio not only provides a larger area for mass transfer passage and facilitates removal of the products, but also promotes higher methanol crossover. The hot-pressed diffusion layer (DL) can mitigate methanol permeation while the non-bonded variant is able to enhance product removal. The increase of membrane thickness helps obtain a lower methanol crossover rate and higher methanol utilisation efficiency, but also depresses cell performance under certain conditions. In this research, the maximum power density of 10.7mWcm−2 is obtained by selecting the current collector with a lower open ratio, the non-bonded DL, and the Nafion 117 membrane. The effect of methanol concentration on the performance of DMFCs is also explored.