Lutein and zeaxanthin have various activities such as anti-age-related macular degeneration, anticataract, anticancer and cardiovascular diseases risk lowering, etc.; however, few studies have been reported on the role of free hydroxyl groups in the antitumor effects of lutein and zeaxanthin. The structure-activity relationship (SAR) of lutein and zeaxanthin di-acetylation derivatives has been investigated. The lutein and zeaxanthin were purified from corn protein residues and structurally modified by di-acetylation, which was characterized with UV/visible and HPLC/MS spectroscopy. The anti-proliferative effects of di-acetylated lutein or zeaxanthin on the human mouth epithelial cancer line KB cell were compared with controls of their original counterparts. Results showed that the anti-tumor activities of the di-acetylation of lutein and di-acetylation of zeaxanthin decreased significantly (p<0.05) at 50 micromol/L. Also the related IC50 dose was increased after di-acetylation. It was suggested that the hydroxyl groups contribute to the anti-tumor activity of lutein and zeaxanthin.