Aims To determine whether the carotenoid production improves stress tolerance of lactic acid bacteria, the cloned enterococcal carotenoid biosynthesis genes were expressed in Lactococcus lactis ssp. cremoris MG1363, and the survival rate of carotenoid-producing engineered MG1363 strain under stress condition was investigated. Methods and Results We cloned carotenoid biosynthesis genes from yellow-pigmented Enterococcus gilvus. The cloned genes consisted of crtN and crtM and its promoter region were inserted into the shuttle vector pRH100, and the resulting plasmid was named pRC. The cloned crtNM was expressed using pRC in noncarotenoid-producing L.lactis ssp. cremoris MG1363. The expression of crtNM led to the production of C30 carotenoid 4,4-diaponeurosporene. After exposure to 32mmoll1 H2O2, low pH (1.5, acidified with HCl), 20% bile acid and 12 mg ml1 lysozyme, the survival rates of the MG1363 strain harbouring pRC were 18.7-, 6.8-, 8.8- and 4.4-fold higher, respectively, than those of MG1363 strain harbouring the empty vector pRH100. Conclusions The expression of carotenoid biosynthesis genes from Ent.gilvus improves the multistress tolerance of L.lactis. Significance and Impact of the study First report of the improvement of multistress tolerance of lactic acid bacteria by the introduction of genes for carotenoid production.