<P><B>Abstract</B></P> <P>Glucose and xylose co-fermentation is crucial to maximize hydrogen yield from waste lignocellulose. In this study, cell growth, sugar consumption, and hydrogen production profiles of <I>Thermoanaerobacter thermosaccharolyticum</I> W16 feeding with a range of glucose and xylose were experimental investigated coupled with kinetic analysis. Results showed although <I>T. thermosaccharolyticum</I> W16 could use both glucose and xylose for hydrogen production, a maximum cell growth rate of 0.27 g/L/h and hydrogen production rate of 14.53 mmol/L/h was found with glucose as sole substrate, the value was 92.8% and 49.8% higher than using xylose as the only carbon source. Further interpolation analysis and experimental demonstration suggested when glucose content in the mixed substrate higher than 58.2%, the inhibitory effect on xylose utilization was increased, but when glucose concentration fell below 21.7%, its utilization will be subject to a certain degree of feedback inhibition. Coupling experimental results with kinetic analysis in this study provides a powerful evidence to further develop the potential of <I>T. thermosaccharolyticum</I> W16 as a biocatalyst for hydrogen production from lignocellulosic biomass.</P> <P><B>Highlights</B></P> <P> <UL> <LI> <I>T. thermosaccharolyticum</I> W16 could co-utilize glucose and xylose for H<SUB>2</SUB> production. </LI> <LI> Glucose dominant cell synthesis and H<SUB>2</SUB> production of <I>T. thermosaccharolyticum</I> W16. </LI> <LI> Threshold value of glucose to xylose ratio for efficient substrate use was obtained. </LI> </UL> </P>