Green hydrogen production through water splitting at low temperatures is highly desired for hydrogen economy. Herein, we demonstrate an in vitro non‐natural enzymatic pathway to utilize the chemical energy stored in xylooligosaccharides from biomass to split water to produce a nearly theoretical yield of H2 (i.e., ≈9.5 H2 per xylose plus water). This pathway was constructed on the basis of the novel activities of phosphopentomutase catalyzing the conversion of d‐xylose 1‐phosphate into d‐xylose 5‐phosphate and of ribose 5‐phosphate isomerase catalyzing the conversions of d‐xylose 5‐phosphate and d‐xylulose 5‐phosphate. This study suggests that the discovery of novel promiscuous enzyme activities is important to implement complicated biotransformations catalyzed by synthetic enzymatic pathways.

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