Carbon nanotubes (CNTs) have potential as not only electrical materials but also biomedical devices. However, some findings have been reported indicating that the use of CNTs is accompanied by a risk of the development of certain diseases such as pulmonary fibrosis and pleura mesothelioma; and one of the reasons for this risk may be macrophage cell death. In the present study, to elucidate the mechanism of macrophage cell death by CNTs, we focused on biomembrane damage caused by multi-walled CNTs (MWCNTs). When the distribution of MWCNTs in RAW264 cells was observed under a light microscope, MWCNTs were located on the surface of the plasma membrane; and a portion of them seemed to stick into it. The acute cytotoxicity toward RAW264 cells was examined by performing the LDH cytotoxic test, and LDH release was detected after exposure to 100 µg/ml CNT. To examine the physical damage to biomembranes by CNT exposure, we conducted a calcein release assay using calcein-encapsulated liposomes. The results indicated that an increase in the permeability of the lipid bilayer was induced by MWCNTs. The present study thus demonstrated for the first time that a high concentration of MWCNTs was cytotoxic to macrophages and suggested that the direct physical perturbation of biomembranes by MWCNTs plays a role in this activity.