C60 fullerene is a promising material because of its unique physiochemical properties. However, previous studies have reported that colloidal aggregates of C60 (nC60) produce toxicity in fish and human cell cultures. The preparation method of nC60 raises questions as to whether the observed effects stem from fullerenes or from the organic solvents used during the preparation of the suspensions. In this paper, we set out to elucidate the mechanism by which tetrahydrofuran (THF) treatment to enhance the preparation of nC60 leads to cytotoxicity in a mouse macrophage cell line. Our results demonstrate that THF/nC60 but not fullerol or aqueous nC60 generates cellular toxicity through a pathway that involves increased intracellular flux and mitochondrial perturbation in RAW 264.7 cells. Interestingly, the supernatant of the THF/n60 suspension rather than the colloidal fullerene aggregates mimics the cytotoxic effects due to the presence of gamma-butyrolactone and formic acid. Thus, the role of nC60 in the cellular responses is likely not due to the direct effect of the nC60 material surface on the cells but is related to the conversion of THF into a toxic byproduct during preparation of the suspension.