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Nucleosomes are the basic structural units of chromatin formed from DNA and histones, revealing the structural features of DNA's stability as genetic material. Despite advances in the understanding of the canonical principles of nucleosome structural organization, the various possible structural states and their implications for genome function require further in-depth study and analysis. In addition, nucleosomes also have important clinical significance. Nucleosomes as structural–functional units of chromatin control the stability of the eukaryotic genome and regulate DNA processes such as transcription, replication, and repair. The influence of nucleosome structure and dynamics produces superior regulation of the expression of related genes and downstream pathways; disturbance and failure of these processes lead to the emergence of various tumor and metabolic diseases. Intervention at the nucleosome level can change the process of diseases earlier and is a feasible option for future diagnosis and treatment. For example, post-translational modifications of nucleosome histones are the most important components of epigenetic regulation systems, and disorder of epigenetic regulation is one of the most important mechanisms of carcinogenesis. Additionally, many nucleosome-binding proteins, such as FACT, PARPs, HDAC, etc., are promising targets for targeted therapy. In this issue, we invite all scientists involved in practical research on nucleosome-binding proteins, various epigenetic factors, and inhibitors to cooperate. This Special Issue aims to collect articles on nucleosomes’ structure and function and their clinical applications.