beta-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 yeastSaccharomyces cerevisiaeto produce beta-carotene from xylose, the second most abundant and inedible sugar component of lignocellulose biomass. Specifically, a beta-carotene biosynthetic pathway containingcrtYB, crtI, andcrtEfromXanthophyllomyces dendrorhouswas introduced into a xylose-fermentingS. cerevisiae. The resulting strain produced beta-carotene from xylose at a titer threefold higher than from glucose. Interestingly, overexpression of tHMG1, which has been reported as a critical genetic perturbation to enhance metabolic fluxes in the mevalonate pathway and beta-carotene production in yeast when glucose is used, did not further improve the production of beta-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 beta-carotene production to be a more respiratory feature of xylose consumption, a larger cytosolic acetyl-CoA pool, and an upregulated expression level of rate-limiting genes in the beta-carotene-producing pathway, includingACS1andHMG1. As a result, 772.8 mg/L of beta-carotene was obtained in a fed-batch bioreactor culture with xylose feeding. Considering the inevitable large scale production of xylose when cellulosic biomass-based bioeconomy is implemented, our results suggest xylose utilization is a promising strategy for overproduction of carotenoids and other isoprenoids in engineeredS. cerevisiae.