<p>Xylitol, a five-carbon sugar alcohol, is recognized as a desirable sugar alternative due to its low-calorie content and metabolism independent of insulin. Its commercial production generally involves the chemical hydrogenation of D-xylose, an approach that is energy-demanding and environmentally unfriendly. Although fermentation offers a biological alternative, it often suffers from low conversion efficiency and limited yields. However, xylitol is an intermediate metabolite in various microbial species, and its biosynthesis can be enhanced through metabolic engineering. Genetically modifying microbial cell factories—such as bacteria, fungi, and yeast—has shown significant improvements in xylitol production. Furthermore, the precursor xylose, which is utilized by microbes, can be derived from lignocellulosic biomass through hydrolysis, offering a more sustainable and cost-effective production route. This review discusses recent advances in the bioproduction of xylitol and highlights various metabolic engineering strategies employed to enhance xylitol yield in microbial cell factories.</p> Graphical Abstract <p></p>