α-Glucosidase inhibitors are used therapeutically to treat type-2 diabetes mellitus. Through a bioassay-guided fractionation technique, three carotenoids, (all-E)-lutein, (all-E)-zeaxanthin and (9-Z)-zeaxanthin, were purified from the green alga Chlorella ellipsoidea, in which (all-E)-lutein and (9-Z)-zeaxanthin had potent α-glucosidase inhibitory activity. IC50 values of (all-E)-lutein and (9-Z)-zeaxanthin were 70 and 53.5 μmol L−1 against Saccharomyces cerevisiae α-glucosidase, respectively, with non-competitive inhibition. In addition, IC50 values of (9-Z)-zeaxanthin against Bacillus stearothermophilus and rat-intestinal α-glucosidase were 805.1 and 671.2 μmol L−1, respectively. The Ki values of (all-E)-lutein and (9-Z)-zeaxanthin against S. cerevisiae α-glucosidase were 78.1 and 16.5 μmol L−1, respectively. Therefore, C. ellipsoidea carotenoids might be utilized as a novel candidate to prevent type-2 diabetes mellitus related disorders in food and medical industry.