Objective To investigate the regularity of chronopharmacology of bruceolic oil emulsion ( BOE) in treating H22 tumor-bearing mice. Methods The KM mice model bearing H22 hepatocellular carcinoma was established. And then the model mice were divided into groups according to the condition of illumination and medicine intervention. After treatment for 10 days, the tumor-growth inhibitory rate, viscera indexes, and hepatic and renal function indexes were observed. Results Tumor mass and ratio of tumor mass to body mass were decreased in medication groups under the normal and disordered illumination. The tumor mass and ratio in mice treated with BOE before dark phase of normal illumination and disordered illumination, and during the dark phase of disordered illumination differed from those of the model group at the corresponding illumination phase (P<0.05) . Under normal illumination, BOE-treated mice at 10 pm of dark phase had lower liver index but higher thymus index than the model group (P < 0.05) . Under disordered illumination, BOE-treated mice at 7 am of dark phase had higher spleen index than the model group ( P < 0.05) . Conclusion BOE exerts certain inhibition of H22 hepatocellular carcinoma under both normal illumination and disordered illumination, and the effect will be better when medication is given in dark phase period.