Beta-carotene is known to exhibit a number of pharmacological and nutraceutical benefits to human health. Metabolic engineering of beta-carotene biosynthesis in Saccharomyces cerevisiae has been attracting the interest of many researchers. A previous work has shown that S. cerevisiae successfully integrated with phytoene synthase (crtYB) and phytoene desaturase (crtI) from Xanthophyllomyces dendrorhous could produce beta-carotene. In the present study, we achieved around 200% improvement in beta-carotene production in S. cerevisiae through specific site optimization of crtI and crtYB, in which five codons of crtI and eight codons of crtYB were rationally mutated. Furthermore, the effects of the truncated HMG-CoA reductase (tHMG1) from S. cerevisiae and HMG-CoA reductase (mva) from Staphylococcus aureus on the production of beta-carotene in S. cerevisiae were also evaluated. Our results indicated that mva from a prokaryotic organism might be more effective than tHMG1 for beta-carotene production in S. cerevisiae.