Xylitol is a natural five-carbon sugar alcohol with potential for use in food and pharmaceutical industries owing to its insulin-independent metabolic regulation, tooth rehardening, anti-carcinogenic, and anti-inflammatory, as well as osteoporosis and ear infections preventing activities. Chemical and biosynthetic routes using D-xylose, glucose, or biomass hydrolysate as raw materials can produce xylitol. Among these methods, microbial production of xylitol has received significant attention due to its wide substrate availability, easy to operate, and eco-friendly nature, in contrast with high-energy consuming and environmental-polluting chemical method. Though great advances have been made in recent years for the biosynthesis of xylitol from xylose, glucose, and biomass hydrolysate, and the yield and productivity of xylitol are substantially improved by metabolic engineering and optimizing key metabolic pathway parameters, it is still far away from industrial-scale biosynthesis of xylitol. In contrary, the chemical synthesis of xylitol from xylose remains the dominant route. Economic and highly efficient xylitol biosynthetic strategies from an abundantly available raw material (i.e., glucose) by engineered microorganisms are on the hard way to forwarding. However, synthetic biology appears as a novel and promising approach to develop a super yeast strain for industrial production of xylitol from glucose. After a brief overview of chemical-based xylitol production, we critically analyzed and comprehensively summarized the major metabolic strategies used for the enhanced biosynthesis of xylitol in this review. Towards the end, the study is wrapped up with current challenges, concluding remarks, and future prospects for designing an industrial yeast strain for xylitol biosynthesis from glucose.