Transferring the recombinant S. cerevisiae T73-63 from 30 oC to 4 oC resulted in 41.4% increment of β-carotene concentration (3.96mg/g dry cell weight) relative to that of 30 oC, which was accompanied with the accumulation of fatty acid and ergosterol. The comparisons of the transcriptional levels of mevalonate pathway genes indicated that the expressions of HMG1, ERG9, ERG19, ERG20 and IDI1 at 4 oC were all higher than those of 30 oC, respectively. This suggested that increased transcriptions of mevalonate pathway genes contribute to the improvement of β-carotene production at low temperature. We also found that supplementation of 30mg/L triclosan, an inhibitor of fatty acid synthesis, led to further 28.3% enhancement of β-carotene concentration (4.94mg/g DCW), which was 18.8% higher than that of 30 oC with the same concentration of triclosan. The higher expressional levels of HMG, ERG19 and ERG20 and the simultaneous increment of ergosterol content (17.8%) suggested that more carbon source was transferred from fatty acid synthesis to mevalonate pathway under the circumstance of appropriately blocking fatty acid synthesis at low temperature (4 oC), which resulted in a higher increment of β-carotene production compared to that of 30 oC. The results of this study collectively suggest that the combination of reducing temperature and adding fatty acid synthesis inhibitors is a potential approach to improve the production of desirable isoprenoid compounds such as carotenoids.