Microbial conversion of plant biomass to value-added products is an attractive option to address the impacts of petroleum dependency. In this study, a bacterial system was developed that can hydrolyze xylan and utilize xylan-derived xylose for growth and production of polyhydroxyalkanoates (PHAs). A β-xylosidase and an endoxylanase were engineered into a P(LA-co-3HB)-producing Escherichia coli strain to obtain a xylanolytic strain. Although PHA production yields using xylan as sole carbon source were minimal, when the xylan-based media was supplemented with a single sugar (xylose or arabinose) to permit the accumulation of xylan-derived xylose in the media, PHA production yields increased up to 18-fold when compared to xylan-based production, and increased by 37 % when compared to production from single sugar sources alone. ¹H-Nuclear magnetic resonance (NMR) analysis shows higher accumulation of xylan-derived xylose in the media when xylan was supplemented with arabinose to prevent xylose uptake by catabolite repression. ¹H-NMR, gel permeation chromatography, and differential scanning calorimetry analyses corroborate that the polymers maintain physical properties regardless of the carbon source. This study demonstrates that accumulation of biomass-derived sugars in the media prior to their uptake by microbes is an important aspect to enhance PHA production when using plant biomass as feedstock.