D-Xylitol is a naturally occurring sugar alcohol present in diverse plants that is used as an alternative sweetener based on a sweetness similar to sucrose and several health benefits compared to conventional sugar. However, current industrial methods for D-xylitol production are based on chemical hydrogenation of D-xylose, which is energy-intensive and environmentally harmful. However, efficient conversion of L-arabinose as an additional highly abundant pentose in lignocellulosic materials holds great potential to broaden the range of applicable feedstocks. Both pentoses D-xylose and L-arabinose are converted to D-xylitol as a common metabolic intermediate in the native fungal pentose catabolism.To engineer a strain capable of accumulating D-xylitol from arabinan-rich agricultural residues, pentose catabolism was stopped in the ascomycete filamentous fungus Aspergillus niger at the stage of D-xylitol by knocking out three genes encoding enzymes involved in D-xylitol degradation (ΔxdhA, ΔsdhA, ΔxkiA). Additionally, to facilitate its secretion into the medium, an aquaglyceroporin from Saccharomyces cerevisiae was tested. In S. cerevisiae, Fps1 is known to passively transport glycerol and is regulated to convey osmotic stress tolerance but also exhibits the ability to transport other polyols such as D-xylitol. Thus, a constitutively open version of this transporter was introduced into A. niger, controlled by multiple promoters with varying expression strengths. The strain expressing the transporter under control of the PtvdA promoter in the background of the pentose catabolism-deficient triple knock-out yielded the most favorable outcome, producing up to 45% D-xylitol from L-arabinose in culture supernatants, while displaying minimal side effects during osmotic stress. Due to its additional ability to extract D-xylose and L-arabinose from lignocellulosic material via the production of highly active pectinases and hemicellulases, A. niger emerges as an ideal candidate cell factory for D-xylitol production from lignocellulosic biomasses rich in both pentoses.In summary, we are showing for the first time an efficient biosynthesis of D-xylitol from L-arabinose utilizing a filamentous ascomycete fungus. This broadens the potential resources to include also arabinan-rich agricultural waste streams like sugar beet pulp and could thus help to make alternative sweetener production more environmentally friendly and cost-effective.