In this study, the materials of polymethyl methacrylate (PMMA) with hydrophilic properties and polydimethylsiloxane (PDMS) with hydrophobic properties were used to form the anode cannels of direct methanol fuel cells (DMFCs). The channels of the DMFCs were fabricated through a microelectromechanical system (MEMS) fabrication process. Two DMFCs were stacked together with a common cathode channel to reduce the volume of the cell and form a twin stack DMFC. The performance of the twin stack DMFC was investigated under different operating conditions, including operation temperature and flow rate. The twin stack DMFC with an anode channel made of PDMS possessed better performance under any specific operating conditions tested in comparison to the DMFC with PMMA anode channel. However, an increase in operation temperature and fuel flow rate resulted in decreased a hydro-resistance reduction effect of PDMS and performance enhancement of the PDMS DMFC was not obvious in high temperature and high flow rate operating conditions. For the PDMS DMFC, the maximum power densities of the stack can be enhanced 70∼80% in comparison to single cells under tested operating conditions.