Epoxyeicosatrienoic acids (EETs), which exert multiple endogenous protective effects, are hydrolyzed into less active dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). However, commercial drugs related to EETs or sEH are not yet in clinical use.Firstly, the plasma concentration of EETs and DHETs of 316 patients with heart failure (HF) were detected and quantitated by liquid chromatography-tandem mass spectrometry. Then, transverse aortic constriction (TAC)-induced HF was introduced in cardiomyocyte-specific Ephx2-/- mice. Moreover, Western blot, real-time PCR, luciferase reporter, ChIP assays were employed to explore the underlying mechanism. Finally, multiple sEH inhibitors were designed, synthesized, and validated in vitro and in vivo.The ratios of DHETs/EETs were increased in the plasma from patients with HF. Meanwhile, the expression of sEH was upregulated in the heart of patients and mice with HF, especially in cardiomyocytes. Cardiomyocyte-specific Ephx2-/- mice ameliorated cardiac dysfunction induced by TAC. Consistently, Ephx2 knockdown protected Angiotensin II (AngII)-treated cardiomyocytes via increasing EETs in vitro. Mechanistically, AngII could enhance the expression of transcript factor Krüppel-like factor 15 (KLF15), which in turn upregulated sEH. Importantly, glimepiride was identified as a novel sEH inhibitor, which benefited from the elevated EETs during HF.Glimepiride attenuates HF in mice in part by increasing EETs.NCT03461107 (https://clinicaltrials.gov).Copyright © 2023. Published by Elsevier Ltd.