AbstractA variety of fuels can be utilized to achieve an extraordinarily high energy density in enzymatic fuel cells (EFCs), including glucose, fructose, and sucrose or maltodextrin composed of hexoses. However, xylose, which is the most abundant pentose, has hardly been exploited for bioelectricity generation because of its slow utilization rate and few xylose metabolic pathways in nature. Here, we demonstrate a reconstituted pentose phosphate pathway in vitro composed of 14 enzymes that completely oxidizes xylose. Either ATP or polyphosphate can be used as a phosphate donor in this system. The Faraday efficiency from xylose to electrons by this pathway was as high as 97.0 %, corresponding to the generation of nearly 20 electrons per molecule of xylose. In the presence of 20 mm xylose, an air‐breathing EFC based on the synthetic pathway generated a power density of 0.36 mW cm−2, much higher than that generated by other xylose‐fed bioelectrochemical systems. These results suggest the great potential of the co‐utilization of hexoses and pentoses from renewable abundant biomass for high‐yield bioelectricity generation in the near future.