The cytotoxicities of three aconitum alkaloids- aconitine, hypaconitine and mesaconitine, and their abilities to bind DNA have been explored. Rat myocardial cells H9c2 were treated with aconitum alkaloids and assessed the cytotoxicities by using MTT assay and flow cytometry. Apoptosis was evidenced by the results of the annexin V/propidium iodide (PI) assay. Aconitine was found to be the most toxic in rat myocardial cells H9c2 in three aconitum alkaloids. At the same time, DNA adducts were isolated and then analyzed by UV-Vis spectroscopy after exposure to alkaloids, which indicated that three alkaloids could bind to DNA in rat myocardial cells H9c2. Furthermore, their binding modes were investigated by UV-Visible, fluorescence, DNA melting studies and ionic strength effect. Results indicated that the interaction between three alkaloids and DNA were intercalation coupled with electrostatic effect. The estimated binding constants were between 4.83 × 105 M-1 to 9.85 × 105 M-1 for three alkaloids at 298 K.