Trihydroxybenzoic acid dimmer is an anti-tumor compound which is separated from water-caltrop. This study is aimed to investigate its anti-proliferation effect on HL-60 cells and the possible mechanism of inducing apoptosis. MTT assay was used to test HL-60 cells proliferation. The apoptosis, ROS levels and the mitochondrial membrane potential were detected by flow cytometry. Cytochrome c released from mitochondria to cytosol fraction was detected by Western blotting. The activity of Caspase-9 and Caspase-3 were measured by colorimetric method. It was found to inhibit HL-60 cells proliferation in a dose and time-dependent manner. Compared with control group, it caused the increase of ROS levels and a concomitant dissipation of the mitochondrial membrane potential and induced cytosolic accumulation of cytochrome c and activities Caspase-9 and Caspase-3. Therefore it can be concluded that mitochondrial-dependent pathways was involved in its induction of apoptosis of HL-60 cells.