In the present study, 15 derivatives of protoberberine (3-17) along with berberine (1) and jatrorrhizine (2) were evaluated for their antineoplastic activities against acute lymphoid leukemia cells (including Reh and Nalm-6 cells) in terms of proliferation inhibition. Compounds 4 (9-bromoethylberberine), 5 (9-chloroethylberberine) and 6 (9-bromopropylberberine) showed most significant inhibitory activities with IC50 values of 0.45, 0.39, and 0.57 μmol/L against Reh cells, while Nalm-6 cells were less sensitive to 4,5,6, with IC50 values of 3.6, 4.3 and 1.17 μmol/L, respectively, which were both stronger than that of the lead compound berberine (1) and jatrorrhizine (2) (> 20 μmol/L, respectively). Furthermore, 4 and 5 could induce apoptosis in acute lymphoid leukemia cells as evidenced by cleavage of PARP in a dosedependent manner, decrease of procaspase-3, increase of active caspase-3 and increase of the levels of cytochrome c in cytoplasm. Moreover, the Reh cells treated with 4 and 5 at the concentration of 0.5 μmol/L for 36 h could significantly lead to the down regulation of β-catenin, and the result demonstrated that the mechanism of the derivatives on tumor were partially involved in the Wnt/β-catenin signal pathway. A PAMPA permeability study of 1, 4, 5, 7 and 11 suggested that side chain substituted derivatives in 9-position could improve the membrane permeability of berberine. It will be helpful for application in vivo assay. These findings suggest that these derivertives may be considered for future studies as promising therapeutic candidate for acute lymphoid leukemia.