SET domain bifurcated 1 (SETDB1), a histone H3K9-specific methyltransferase, is crucial for heterochromatin formation and intestinal homeostasis, but its role in intestinal ischemia-reperfusion injury (IRI) remains unclear. This study investigated changes in SETDB1-mediated nuclear chromatin regulation in intestinal epithelial cells (IECs) using an IRI mouse model. Jejunal samples were collected after 75 min of ischemia followed by 24 hr of reperfusion. Sinefungin was administered as a histone methyltransferase inhibitor. Morphologic changes were evaluated using hematoxylin-eosin staining and electron microscopy, and cell-adhesion molecule expression, including ZO-1, E-cadherin, integrin-β4, and laminin, was evaluated using immunohistochemistry. Super-resolution microscopy analyzed intranuclear SETDB1 localization and heterochromatin formation in IECs. IRI-affected jejunum exhibited massive IEC detachment, dilated intercellular spaces, basement membrane damage, and decreased expression of E-cadherin and integrin-β4. Sinefungin prevented these changes, however. The proportion of IECs expressing nuclear SETDB1 throughout the euchromatin was significantly higher in IRI-affected jejunum (77.8%) than sham-treated (3.0%) or sinefungin-treated, IRI-affected jejunum (2.7%). The proportion of IECs with decreased heterochromatin was significantly higher in sinefungin-treated, IRI-affected jejunum (84.3%) than untreated IRI-affected jejunum (15.6%). These findings suggest that SETDB1-mediated chromatin regulation is pivotal in intestinal IRI and represents a potential therapeutic target.