Chromosomes consist of various domains with different transcriptional activities separated by chromatin boundary sequences such as insulator sequences. Recent studies suggested that CTCF or other chromatin loop-forming protein binding sequences represented typical insulators. Alternatively, some long nucleosome-excluding DNA sequences were also reported to exhibit insulator activities in yeast and sea urchin chromosomes, although specific binding of loop-forming proteins was not expected for them. However, the mechanism of the insulator activities of these sequences and the possibilities of similar insulators existing in other organisms remained unclear. In this study, we first constructed and performed simulations of a coarse-grained chromatin model containing nucleosome-rich and nucleosome-excluding DNA regions. We found that a long nucleosome-excluding region between two nucleosome-rich regions could markedly hinder the associations of two neighboring chromatin regions owing to the stronger long-term-averaged rigidity of the nucleosome-excluding region compared to that of nucleosome-rich regions. Subsequent analysis of the genome-wide nucleosome positioning, protein binding, and DNA rigidity in human cells revealed that some nucleosome-excluding rigid DNA sequences without bound chromatin looping proteins could exhibit insulator activities, functioning as chromatin boundaries in various regions of human chromosomes.