With the rapid development in the medical application of carbon nanomaterials such as carbon nanohorns (CNHs), carbon nanotubes, and graphene based nanomaterials, understanding the long-term fate, health impact, excretion, and degradation of these materials have become crucial. Here, the in vitro biodegradation of CNHs was determined using a non-cellular enzymatic oxidation method and two types of macrophage cell lines. Approximately 60% of the CNHs were degraded within 24 h in a phosphate buffer solution containing myeloperoxidase. Furthermore, approximately 30% of the CNHs were degraded by both RAW 264.7 and THP-1 macrophage cells within 9 days. Inflammation markers such as pro-inflammatory cytokines interleukin 6 and tumor necrosis factor α were not induced by exposure to CNHs. However, reactive oxygen species were generated by the macrophage cells after uptake of CNHs, suggesting that these species were actively involved in the degradation of the nanomaterials rather than in an  inflammatory pathway induction.