Abstract: HPLC was applied to the determination of sitosterol in vegetable oil. The extraction conditions of additional sitosterol in the mixed oil were optimized as follows: the extraction times of 4 times; the centrifugal speed of 2 000 r·min-1; the saponification temperature at 80 ℃ for 3.5 h. C18 reverse-phase chromatographic column was used as stationary phase and a mixture of methanol and water (98.5+1.5) was used as mobile phase. UV-detection was made at the wavelength of 210 nm. Linear relationship between values of peak area and mass concentration of sitosterol was kept in the range of 12.5-1 000 mg·L-1 with detection limit (3S/N) of 5 mg·L-1. Values of recovery found by standard addition method were in the range of 86.2%-98.7%, and values of RSD′s (n=6) in the range of 2.4%-3.9%.