β-carotene is a natural pigment and health-promoting metabolite, and has been widely used in the nutraceutical, feed and cosmetic industries. Here, we engineered a GRAS yeast Saccharomyces cerevisiae to produce β-carotene from xylose, the second most abundant and inedible sugar component of lignocellulose biomass. Specifically, a β-carotene biosynthetic pathway containing crtYB, crtI and crtE from Xanthophyllomyces dendrorhous were introduced into a xylose-fermenting S. cerevisiae. The resulting strain produced β-carotene from xylose at a titer three-fold higher than from glucose. Interestingly, overexpression of tHMG1, which has been reported as a critical genetic perturbation to enhance metabolic fluxes in the mevalonate (MVA) pathway and β-carotene production in yeast when glucose is used, did not further improve the production of β-carotene from xylose. Through fermentation profiling, metabolites analysis and transcriptional studies, we found the advantages of using xylose as a carbon source instead of glucose for β-carotene production to be a more respiratory feature of xylose consumption, a larger cytosolic acetyl-CoA pool, and up-regulated expression levels of rate-limiting genes in the β-carotene producing pathway, including ACS1 and HMG1. As a result, 772.81 mg/L of β-carotene was obtained in a fed-batch bioreactor culture with xylose feeding. Considering the inevitable production of xylose at large scales when cellulosic biomass-based bioeconomy is implemented, our results suggest xylose utilization is a promising strategy for overproduction of carotenoids and other isoprenoids in engineered S. cerevisiae.