Lutein is a kind of natural carotenoids possessing many pharmacological effects. The application of lutein was limited mainly due to its low oral bioavailability caused by poor aqueous solubility. Nanocrystal formulation of lutein was developed to improve the oral bioavailability in this study. The nanosuspension was prepared by the anti-solvent precipitation-ultrasonication method and optimized by Box-Behnken design, followed by freeze-drying to obtain lutein nanocrystals. The nanocrystals were characterized on their physical properties, in vitro dissolution and in vivo absorption performance. Lutein nanocrystals showed as tiny spheres with an average particle size of 110.7 nm. The result of diffractograms indicated that the percent crystallinity of lutein was 89.4% in coarse powder and then declined in nanocrystal formulation. The saturated solubility of lutein in water increased from 7.3 μg/ml for coarse powder up to 215.7 μg/ml for lutein nanocrystals. The dissolution rate of lutein nanocrystals was significantly higher than that of coarse powder or the physical mixture. The C and AUC of lutein nanocrystals after oral administration in rats was 3.24 and 2.28 times higher than those of lutein suspension, respectively. These results indicated that the nanocrystal formulation could significantly enhance the dissolution and absorption of lutein and might be a promising approach for improving its oral bioavailability.