Harvesting electricity from microbial electron transfer is believed as a promising way of renewable energy generation. However, a major challenge lies in the still-unknown mechanisms of extracellular electron transfer, especially how microbes consume electrons from the cathode to catalyze oxygen reduction. Here we report a previously undescribed yet significant extracellular electron uptake pathway mediated by inevitably produced H2O2, contributing up to 45% of the total biocurrent. This new H2O2-based bioelectrochemical respiration depends on the continuous supply of electrons from the electrode and the presence of the catalase katG. Selective enhancement of two-electron oxygen reduction on the cathode results in a 2.4-fold increase in biocurrent, and both autotrophic biosynthesis and energy production pathways are upregulated to sustain the H2O2-based respiration. Our results highlight the importance of two-electron oxygen reduction in bioelectron uptake at the cathode and provide a basis for the design of bioelectricity production systems.