Berberine (BBR), a major active component of natural plant product, has recently been demonstrated to be clinically effective in alleviating type 2 diabetes mellitus (T2DM). In this study, BBR and 8-alkylberberine were successively halogenated with bromine, iodine, chlorine under various conditions. HepG2 cell lines, phenotypically similar to human hepatocytes, were used to study the effect of the BBR derivatives on glucose consumption (GC) in vitro. The glucose-lowering effect of chloroberberine and bromoberberine was better than non-modified BBR under the concentrations studied (0.5, 1, 2, 4 μg/mL). Compared to the control, GC of HepG2 cells treated with chloroberberine at 1 μg/mL was increased by 90%. 8-Alkylberberine and 8-alkyl-13-bromoberberine did not exhibit any glucose-lowering effect. 8-Octylberberine caused the reduction of GC in a dose-dependent manner. The MTT assay was used to determine the cytotoxicity of BBR derivatives; the cytotoxicity of chloroberberine and iodoberberine was lower than that of the parent BBR. Meanwhile, the inhibition of HepG2 cell proliferation of 8-alkylberberine was stronger than that of 8-alkyl-13-bromoberberine. These observations suggest that chloroberberine could enhance the glucose-lowering activity and protect HepG2 cell lines as well. 8-Octylberberine showed the strongest HepG2 inhibition activity amongst all the synthesized compounds, which was over 36-fold higher than its parent BBR, and thus might be used as an anticancer agent. These results may open up new avenues for therapeutic applications of BBR in the treatment of T2DM.