We propose a computational Molecular Dynamic (MD) study to evaluate the interaction of C₆₀ fullerene in contact with lipid membranes, as a function of the bilayers cholesterol content. Pure 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and DOPC/cholesterol^XX (XX = 10%, 20% and 30%) membranes where investigated. An analysis involving umbrella pulling techniques to force the extraction of DOPC lipid and/or cholesterol molecules in comparison with an insertion of a fullerene C₆₀ molecule into the lipid/cholesterol membranes was performed in order to verify the force/energy required for the C₆₀@DOPC/cholesterol partitioning. In addition, electron spin resonance experiments using a lipid and an amphiphilic spin labels were performed to investigate the DOPC-cholesterol interactions and to mimic the C₆₀ fullerene-membrane interactions. The spectroscopic results corroborated the MD ones and indicated that the presence of cholesterol in the lipid bilayers increased the membrane rigidity as well as the force required to C₆₀ insertion/extraction.