Lycopene epsilon-cyclase (epsilon-LCY) is a key enzyme that catalyzes the synthesis of alpha-branch carotenoids through the cyclization of lycopene. Two cDNA molecules encoding epsilon-LCY (designated Nt epsilon-LCY1 and Nt epsilon-LCY2) were cloned from Nicotiana tabacum. Nt epsilon-LCY1 and Nt epsilon-LCY2 are encoded by two distinct genes with different evolutionary origins, one originating from the tobacco progenitor, Nicotiana sylvestris, and the other originating from Nicotiana tomentosiformis. The two coding regions are 97% identical at the nucleotide level and 95% identical at the amino acid level. Transcripts of Nt epsilon-LCY were detectable in both vegetative and reproductive organs, with a relatively higher level of expression in leaves than in other tissues. Subcellular localization experiments using an Nt epsilon-LCY1-GFP fusion protein demonstrated that mature Nt epsilon-LCY1 protein is localized within the chloroplast in Bright Yellow 2 suspension cells. Under low-temperature and low-irradiation stress, Nt epsilon-LCY transcript levels substantially increased relative to control plants. Tobacco rattle virus (TRV)-mediated silencing of epsilon-LCY in Nicotiana benthamiana resulted in an increase of beta-branch carotenoids and a reduction in the levels of alpha-branch carotenoids. Meanwhile, transcripts of related genes in the carotenoid biosynthetic pathway observably increased, with the exception of beta-OHase in the TRV-epsilon-lcy line. Suppression of epsilon-LCY expression was also found to alleviate photoinhibition of Potosystem II in virus-induced gene silencing (VIGS) plants under low-temperature and low-irradiation stress. Our results provide insight into the regulatory role of epsilon-LCY in plant carotenoid biosynthesis and suggest a role for epsilon-LCY in positively modulating low temperature stress responses.