Annatto and its derivatives are members of carotenoids with long-chain conjugated polyenes, which are widely used as food additives and antioxidant. However, carotenoids can also act as pro-oxidant under certain circumstances. To explore the biochemical behavior of annatto and its derivatives, the DNA damage effects by norbixin to the copper(II) ions mediated DNA damage was evaluated herein. It has been found that norbixin was capable of promoting copper(II) caused DNA damage on supercoiled plasmid DNA, whereas the long-chain saturated system such as lauric acid did not. These DNA damage showed strong dependency on both the concentration of norbixin and the interaction time. For the mechanism of damage promotion by norbixin, the long-chain conjugated polyenes unit may participate in the reductive reaction of copper(II) ion to generate free radical, and gave stronger DNA damage through the interactions between DNA and the radicalized carotenoids. The control experiments showed that the redox cycle between Cu(I) and Cu(II), hydroxyl radical, singlet oxygen and hydrogen peroxide may play essential roles in the cleavage reaction.