Abstract: Based on the established detection method for chiral resolution of aleuritic acid by HPLC-ELSD, the resolution condition of stereoisomerism of aleuritic acid was established by supercritical fluid chromatography(SFC) method and the optimum conditions were as follows:CHIRALPAK AY-H(25 cm×3.0 cm, 5 μm)column, carbon dioxide-alcohol(70:30, volume ratio) as the mobile phase, the flow rate 70 mL/min, column temperature 35℃, detection wavelength 208 nm, sample injection concentration 77.78 g/L, injection volume 3 mL each time. Two compounds(A and B) were prepared by above SFC method. The result indicated that A and B were stereoisomers of aleuritic acid by characterization of FT-IR, ¹H NMR, ¹³C NMR, DSC, TG and XRD as well as by comparison with the authentic samples. Furthermore, A and B compounds were confirmed as enantiomers of aleuritic acid by specific rotation and vibrational circular dichroism(VCD) spectra. According to melting point of synthetic threo and erythro aleuritic acid previously reported, the stereochemical compositions of aleuritic acid extracted by strong alkali saponification process from lac resin were a couple of enantiomers with threo configuration and possibly composed by 9R, 10R, 16-trihydroxyhexadecanoic acid and 9S, 10S, 16-trihydroxyhexadecanoic acid.