In eukaryotic cells, DNA has to bend significantly to pack inside the nucleus. Physical properties of DNA such as bending flexibility and curvature are expected to affect DNA packaging and partially determine the nucleosome positioning patterns inside a cell. DNA CpG methylation, the most common epigenetic modification found in DNA, is known to affect the physical properties of DNA. However, its detailed role in nucleosome formation is less well-established. In this study, we evaluated the effect of defined CpG patterns (unmethylated and methylated) on DNA structure and their respective nucleosome-forming ability. Our results suggest that the addition of CpG dinucleotides, either as a (CG)n stretch or (CGX8)n repeats at 10 bp intervals, lead to reduced hydrodynamic radius and decreased nucleosome-forming ability of DNA. This effect is more predominant for a DNA stretch ((CG)5) located in the middle of a DNA fragment. Methylation of CpG sites, surprisingly, seems to reduce the difference in DNA structure and nucleosome-forming ability among DNA constructs with different CpG patterns. Our results suggest that unmethylated and methylated CpG patterns can play very different roles in regulating the physical properties of DNA. CpG methylation seems to reduce the DNA conformational variations affiliated with defined CpG patterns. Our results can have significant bearings in understanding the nucleosome positioning pattern in living organisms modulated by DNA sequences and epigenetic features. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 517–524, 2014.