This study was purposed to explore the effects of Tanreqing injection on the biologic activities of human acute T lymphoblastic leukemia cell line Molt4 in vitro and its mechanism. Tanreqing injection was proportionally diluted and divided into 9 groups of different concentrations, including 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256 and 1:512. Molt4 cells were treated with those different concentrations of Tanreqing injection, and the cell growth status at various time points of different concentrations was observed under microscope. CCK-8 assay was employed to detect ability of cell proliferation, growth curve was drawn, the inhibition ratio and 50% inhibiting concentration (IC(50)) were calculated. Flow cytometry with PI and PI/Annexin V double stainings were used to detect the cell cycle and apoptosis of Molt4 cells after the treatment of Tanreqing injection respectively. Caspase-3 and Bcl-2 mRNA expression of Molt4 cells were determined by real-time quantitative PCR. The results showed that Tanreqing injection displayed an inhibitory effect on the proliferation of Molt4 cell line. In 1:2, 1:4, 1:8 and 1:16 concentration groups, great cytotoxicity was observed and numerous cells were dead. The inhibitory effect of Tanreqing injection was dose-dependent. IC(50) was 1:142 dilution concentration. In the 1:32 concentration group, S phase cell quantity remarkably decreased (p < 0.05) and apoptosis rate significantly increased (p < 0.05) at 72 hours after Tanreqing injection treatment. Simultaneously, caspase-3 mRNA expression increased and Bcl-2 mRNA expressions was downregulated (p < 0.05). It is concluded that the Tanreqing injection has an inhibitory effect on Molt4 cell proliferation and promotes its apoptosis. These biological effects of Tanreqing injection are partly related to cell reduction in S phase, downregulation of bcl-2 gene and upregulation of caspase 3.