The interest on functionalized fullerenes in the field of nanomedicine has seen a significant increase in the past decade. However, the different methods employed to increase C60 solubility profoundly influence the physicochemical properties and the toxicological effects of these compounds, thus complicating the evaluation of their toxicity and potential therapeutic use. Here we report a whole-transcriptome RNA-seq analysis assessing the effect of two fullerenes (1 and 2) on gene expression in the human MCF7 cell line. Although these two compounds had previously been characterized by in vitro studies as having a cytotoxic and null effect respectively, to date the mechanisms at the basis of this different behavior and, more in general, at the basis of the effect of most fullerene derivatives in living cells are still completely unknown. Our data evidence that: a) fullerene 2 caused a significant, time-dependent alteration of gene expression, whereas 1 only had a negligible effect; b) the biological processes mostly influenced over the 48hours experimental time course were transcription, protein synthesis, cell cycle progression and cell adhesion; c) the gene expression signature of 2-treated cells was strikingly similar to those induced by selective inhibitors of mTOR signaling, thus suggesting an effect on this pathway for fullerene 2. Our work represents the first approach towards the application of RNA-seq to the study of the molecular mechanisms underlying the interaction of fullerenes with cellular systems and provides an objective view of the feasibility and the safety of these nanomaterials for a medical application.