Ischemic arrhythmias are the main causes of sudden cardiac death. It has been reported that soluble epoxide hydrolase inhibitors (sEHis) could prevent arrhythmias; however, the underlying molecular mechanisms remain unclear. In recent years, the proarrhythmic role of microRNA-1 (miR-1) has been investigated. This study aimed to elucidate whether sEHis prevented ischemic arrhythmias by suppressing miR-1. The primary neonatal mouse ventricular myocyte model of miR-1 overexpression was established by incubating with agonist microONTM mmu-miR-1a-3p agomir (DAEDstain^TM Dye) (agomiR-1). The sEHi, trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB), was administered following miR-1 overexpression. Quantitative real-time PCR (qPCR) and western blotting were used to test alterations in the expression of miR-1 and its target mRNAs GJA1 and KCNJ2 and their respective encoded proteins connexin 43 (Cx43) and the K⁺ channel subunit (Kir2.1). The whole-cell patch-clamp technique was used to record the alterations of the inward rectifying K⁺ current (I_K1). Compared with the control group, miR-1 levels were significantly increased in the agomiR-1 group (p < 0.05), which suggested the successful construction of the miR-1 overexpression model. Compared with the control group, the levels of GJA1 and KCNJ2 mRNAs and Cx43 and Kir2.1 proteins in the agomiR-1 group were significantly decreased, and I_K1 was significantly impaired (all p < 0.05). The miR-1 levels were dose-dependently decreased by t-AUCB, whereas t-AUCB dose-dependently increased the levels of GJA1 and KCNJ2 mRNAs and Cx43 and Kir2.1 proteins. Furthermore, t-AUCB restored the impaired I_K1 (all p < 0.05). In conclusion, the sEHi t-AUCB has the ability to down-regulate proarrhythmic miR-1 and up-regulate its target genes and proteins, eventually restoring I_K1.