Epimedium-derived flavonoid glycosides are widely used for the prevention of osteoporosis, but these compounds generally exhibit poor membrane permeability and oral absorption. To address these limitations, the bioactive lipophilic aglycone icaritin (ICT) was selected and successfully developed into nanocrystals (ICTN) through the antisolvent-precipitation method. After the parameters in the preparation of ICTN were optimized, the morphology, crystallinity, adsorption of the stabilizers on the ICT surface, and the dissolution of the resulting nanocrystals were characterized. The pharmacokinetics in rat and the in vitro antiosteoporosis activity of serum withdrawn after the oral administration of ICTN to rats on mouse osteoblastic cells were evaluated. Consistent with its good performance in stabilizing the ICT nanosuspension, atomic force microscopy showed that hydroxypropyl methylcellulose (HPMC) exhibits better adsorption on the ICT surface compared with other stabilizers. Needle-shaped crystals (∼220 nm in diameter) with a high drug loading (∼90%) were generated when 0.16 mL of the ICT acetone solution (10 mg/mL) was injected quickly into 2 mL of the HPMC solution (0.02%, w/w) under ultrasonication for 10 s at room temperature. The thermal analysis demonstrated that the majority of the particles are in their crystalline forms, similarly to the unformulated ICT. After oral administration, ICTN exhibited a faster dissolution rate and significantly faster absorption, as supported by the increased AUC and C and the reduced T of these nanocrystals compared with the raw suspension (p < 0.05). Compared with blank serum, enhanced proliferation and differentiation activities were observed when serum withdrawn after the oral administration of ICTN in rat was incubated with osteoblast MC3T3-E1 cells. The present delivery system could provide a new promising strategy for BCS IV glycoside of flavonoids or other natural products by formulation of their bioactive lipophilic aglycone forms to enhance oral absorption and in vivo bioactivity. © 2013 American Chemical Society.