Lycopene ϵ-cyclase (LCY-ϵ) is involved in the first step of the α-branch synthesis pathway of carotenoids from lycopene in plants. In this study, to enhance carotenoid synthesis via the β-branch-specific pathway [which yields β-carotene and abscisic acid (ABA)] in sweetpotato, the expression of IbLCY-ϵ was downregulated by RNAi (RNA interference) technology. The RNAi-IbLCY-ϵ vector was constructed using a partial cDNA of sweetpotato LCY-ϵ isolated from the storage root and introduced into cultured sweetpotato cells by Agrobacterium-mediated transformation. Both semi-quantitative Reverse transcription polymerase chain reaction (RT-PCR) of carotenoid biosynthesis genes and high-performance liquid chromatography (HPLC) analysis of the metabolites in transgenic calli, in which the LCY-ϵ gene was silenced, showed the activation of β-branch carotenoids and its related genes. In the transgenic calli, the β-carotene content was approximately 21-fold higher than in control calli, whereas the lutein content of the transgenic calli was reduced to levels undetectable by HPLC. Similarly, expression of the RNAi-IbLCY-ϵ transgene resulted in a twofold increase in ABA content compared to control calli. The transgenic calli showed significant tolerance of 200 mM NaCl. Furthermore, both the β-branch carotenoids content and the expression levels of various branch-specific genes were higher under salt stress than in control calli. These results suggest that, in sweetpotato, downregulation of the ϵ-cyclization of lycopene increases carotenoid synthesis via the β-branch-specific pathway and may positively regulate cellular defenses against salt-mediated oxidative stress.