It is unknown how the composition and structure of DNA within the cell affect spontaneous mutations. Theory suggests that in eukaryotic genomes, nucleosomal DNA undergoes fewer C→T mutations because of suppressed cytosine hydrolytic deamination relative to nucleosome-depleted DNA. Comparative genomic analyses and a mutation accumulation experiment showed that nucleosome occupancy nearly eliminated cytosine deamination, resulting in an ~50% decrease of the C→T mutation rate in nucleosomal DNA. Furthermore, the rates of G→T and A→T mutations were also about twofold suppressed by nucleosomes. On the basis of these results, we conclude that nucleosome-dependent mutation spectra affect eukaryotic genome structure and evolution and may have implications for understanding the origin of mutations in cancers and in induced pluripotent stem cells.