Stability of microencapsulated marigold lutein under three different storage conditions (dark and vacuum, light and vacuum, air and light) at 4, 20, and 37°C was investigated. Major lutein stereoisomers, including all-trans lutein, 13-cis lutein, 13′-cis lutein, 9-cis lutein, and 9′-cis lutein, were rapidly separated within 12 minutes by reverse-phase C30-HPLC-PDA using methanol, methylene chloride, and acetonitrile as eluent, which could provide more explicit information for identifying and distinguishing stereoisomers. The content of all-trans lutein decreased during storage time for all the treatments, and the degradation of all-trans lutein fitted the first order model as correlation coefficients (R2) were higher than 0.95, microencapsulated marigold lutein with vacuum packed were preserved best in dark and 4°C storage.