The impact of structural parameters and operating conditions has not been researched yet for vapor-fed operation of a DMFC at near-ambient conditions. Thus, a detailed parameter study that included reference cell measurements to assess anode and cathode losses separately was performed. Among other parameters like temperature or air stoichiometry, different opening ratios that controlled evaporation of methanol into the vapor chamber were examined. Water management was found to be a critical parameter for a vapor-fed DMFC. Depletion of water inside the anode catalyst layer, especially at higher current densities, decreased performance of the fuel cell substantially. Back diffusion of water from the cathode to the anode was examined. A micro-structured cathode electrode that increased water back diffusion due to a reduced mass transfer resistance was developed and investigated. Finally, efficiencies and heat losses of a vapor-fed DMFC were determined.