The SUR2B/Kir6.1 channel openers iptakalim and natakalim reverse cardiac remodeling and ameliorate endothelial dysfunction by re-establishing the balance between the nitric oxide and endothelin systems. In this study, we investigated the microRNAs (miRs) involved in the molecular mechanisms of SUR2B/Kir6.1 channel opening in chronic heart failure. Both iptakalim and natakalim significantly upregulated the expression of miR-1-3p, suggesting that this miR is closely associated with the therapeutic effects against chronic heart failure. Bioinformatic analysis showed that many of the 183 target genes of miR-1-3p are involved in cardiovascular diseases, suggesting that miR-1-3p plays a vital role in such diseases and vascular remodeling. Target gene prediction showed that miR-1-3p combines with the 3′ untranslated region (UTR) of endothelin-1 (ET-1) mRNA. Iptakalim and natakalim upregulated miR-1-3p expression and downregulated ET-1 mRNA expression in vitro. The dual luciferase assay confirmed that there is a complementary binding sequence between miR-1-3p and the 3′ UTR 158–165 sequence of ET-1 mRNA. To verify the effect of miR-1-3p on ET-1, lentiviral vectors overexpressing or inhibiting miR-1-3p were constructed for the transduction of rat primary cardiac microvascular endothelial cells. The results showed that natakalim enhanced the miR-1-3p level. miR-1-3p overexpression downregulated the expression of ET-1, whereas miR-1-3p inhibition had the opposite effect. Therefore, we verified that SUR2B/Kir6.1 channel openers could correct endothelial imbalance and ameliorate chronic heart failure through the miR-1-3p/ET-1 pathway in endothelial cells. Our study provides comprehensive insights into the molecular mechanisms behind the SUR2B/Kir6.1 channel’s activity against chronic heart failure.