Carbon fullerene (C60) has emerged at the forefront of nanoscale research and application due to its unique properties. As the production of this nanoparticle rapidly increases, it can be released into natural aquatic environments and can accumulate in biological systems. This research examined the effects of humic acid and fetal bovine serum (FBS), which are ubiquitous in aquatic environments and representative of blood plasma in living organisms, respectively, on bioavailability of fullerene. Bioavailability was investigated using in vitro methods for lipid membrane accumulation and cellular uptake studies. Humic acid and FBS significantly changed the characteristics of fullerene including its particle size and surface charge. The effects of humic acid on lipid accumulation of fullerene depended on the lipid head charge. FBS also significantly decreased the lipid accumulation when positively charged and zwitterionic head groups were present on the lipids, possibly due to the higher steric repulsion of the protein coated nanoparticles. In addition, both humic acid and FBS protein effectively lowered the amounts of fullerene taken up by Caco-2 cells, which are derived from a human colorectal adenocarcinoma and have similar functions to the small intestinal epithelium. Results of this study suggest that surface modification of fullerene by environmentally relevant matrices can significantly affect the biological transport, as well as the possible toxicity of this nanomaterial.