Chromatin accessibility is closely associated with chromatin functions such as gene expression, DNA replication, and maintenance of DNA integrity. However, the relationship between chromatin accessibility and plant hormone signaling has remained elusive. Here, based on the correlation between chromatin accessibility and DNA damage, we used the sensitivity to DNA double strand breaks (DSBs) as an indicator of chromatin accessibility and demonstrated that auxin regulates chromatin accessibility through the TIR1/AFBs signaling pathway in proliferative cells. Treatment of proliferating plant cells with an inhibitor of the TIR1/AFBs auxin signaling pathway, PEO-IAA, caused chromatin loosening, indicating that auxin signaling functions to decrease chromatin accessibility. In addition, a transcriptome analysis revealed that several histone H4 genes and a histone chaperone gene, FAS1, are positively regulated through the TIR1/AFBs signaling pathway, suggesting that auxin plays a role in promoting nucleosome assembly. Analysis of the fas1 mutant of Arabidopsis thaliana confirmed that FAS1 is required for the auxin-dependent decrease in chromatin accessibility. These results suggest that the positive regulation of chromatin-related genes mediated by the TIR1/AFBs auxin signaling pathway enhances nucleosome assembly, resulting in decreased chromatin accessibility in proliferative cells.