Although considered as the most viable approach for mobile powers, traditional polymer electrolyte membrane fuel cells require burdensome humidification and water management systems. The bipolar fuel cell (BPFC) or hybrid membrane fuel cell won noticeable interest because its potential self-humidification. BPFC is a novel polyelectrolyte membrane fuel cell involving both anion and cation polyelectrolyte and a new kind of acidic-alkaline bipolar reaction interface was presented in the cell. The potential advantages of a BPFC are twofold: (a) the alkaline cathode with inherently faster kinetics that allows use of non-platinum catalysts, thereby significantly reducing the total cost of a fuel cell; (b) the water generation or dissociation reaction takes place at the bipolar interface providing the possibility to devise self-humidification over the entire cell. Despite the BPFC have such attractive potentials, the BPFC developed to date had not yet demonstrated these feature and have operated at very modest power densities. As the development of BPFC is still in their infancy, it still remains uncertain how the bipolar interface works and effects in the cell. In the present study, four types of bipolar membrane electrode configuration containing two or one kind of bipolar interface, either water generation or dissociation interfaces, were fabricated to evaluate the effect of bipolar interface on the cell output performance. Results show that the preferred configuration with only one bipolar interface generating water can benefit the cell output. The reason could be the faster water formation reaction kinetics in this kind of bipolar interface and lower interfacial potential loss with only one bipolar interface. Within this preferred membrane electrode configuration, we have realized and demonstrated the BPFC that operate under completely self-humidifying conditions for prolonged periods successfully. As we can see, optimization of the membrane electrode configurations and further advances in fabricating bipolar interface would open the way for the development of practical self-regulating portable fuel cells.