The crude ethanol extract of Pulsatilla chinensis (Bunge) Regel roots was extracted successively with light petroleum, dichloromethane and n-butanol. The light petroleum fraction (PEF) exhibited potent anti-proliferation activity on HL60 cells with an IC 50 value of 14 μg/mL. As a result, ICR mice transplanted with tumor strain S180 were employed for testing the effectiveness of drug administration of PEF. The tumor inhibitory rate was 36.7% at a dose of 20 mg/kg/d, which was higher than the positive control, which produced 31.5% inhibition. However, an unusual phenomenon was observed in that the tumor inhibitory response was reverse dose-dependent since tumor inhibition was only 19.5% at a dose of 180 mg/kg/d. GC-MS revealed that the main components of the PEF were C-19 and C-22 polyunsaturated fatty acids. Previous studies had revealed that polyunsaturated fatty acids exhibit either anti-tumor or tumor promoting activities, and so it is proposed that the effects of PEF on tumor growth is dependent on dosage.