AbstractThe role of pescadillo1 (PES1) in regulating vascular permeability has been unknown. This study probes the role of PES1 and its mediated molecular mechanism in modulating vascular hyperpermeability in diabetic mice. Male C57BL/6J anddb/dbmice were fed a standard diet and a ketogenic diet (KD). Meanwhile, mouse vascular endothelial cells (MVECs) were treated with β‐hydroxybutyric acid (β‐HB),Pes1siRNA or aPes1overexpression plasmid. Additionally, knockout (KO) ofPes1in mice was applied. After 12 weeks of feedings, enhanced vascular PES1 expression in diabetic mice was inhibited by the KD. The suppression of PES1 was also observed in β‐HB‐treated MVECs. In mice withPes1KO, the levels of vascular VEGF and PES1 were attenuated, while the levels of vascular VE‐cadherin, Ang‐1 and Occludin were upregulated. Similar outcomes also occurred after the knockdown ofPes1in cultured MVECs, which were opposite to the effects induced by PES1 overexpression in MVECs. In vitro and in vivo experiments showed that high glucose concentration‐induced increases in vascular paracellular permeability declined after MVECs were treated by β‐HB or by knockdown ofPes1. In contrast, increases in vascular permeability were induced by overexpression ofPes1, which were suppressed by coadministration of β‐HB in cultured endothelial cells. Similarly declines in vascular permeability were found byPes1knockdown in diabetic mice. Mechanistically, β‐HB decreased PES1‐facilitated ubiquitination of VE‐cadherin. The KD suppressed the diabetes‐induced increase in PES1, which may result in vascular hyperpermeability through ubiquitination of VE‐cadherin in type 2 diabetic mice.