Previous studies found that piRNAs could participate in disease progression by regulating DNA methylation, but there are few reports on their roles in heart failure (HF).The level of piRNA-6426 in the venous blood of HF patients and volunteers was detected by RT-qPCR. Hypoxia-induced cardiomyocytes were transfected with lentiviral-mediated piRNA-6426 overexpression vector (LV-piRNA-6426) or together with LV-DNMT3B, and then cell viability and apoptosis, glucose uptake, ROS production, LDH activity and secretion of inflammatory factors were detected. Also, cardiomyocytes were transfected with LV-piRNA-6426, sh-piRNA-6426 or sh-SOAT1, as well as LV-piRNA-6426 or together with LV-DNMT3B or sh-DNMT3B. The interaction between piRNA-6426 and methyltransferase 3B (DNMT3B) was detected with RNA immunoprecipitation (RIP). And the methylation level of sterol o-acyltransferase 1 (SOAT1) and the enrichment of DNMT3B in the SOAT1 promoter were detected with Methylation-specific PCR (MSP) and ChIP assays. Then a HF rat model constructed with coronary artery occlusion method was injected with LV-piRNA-6426, and heart function index and infarcted area of rat heart were detected.piRNA-6426 expression was decreased in the blood of HF patients. LV-piRNA-6426 transfection increased the enrichment of DNMT3B in SOAT1 promoter, thereby inhibiting the expression level of SOAT1, and decreased hypoxia-induced oxidative stress and inflammation in cardiomyocytes, while sh-piRNA-6426 transfection had the opposite effect. And LV-DNMT3B transfection enhanced the effect of LV-piRNA-6426 transfection on SOAT1 expression and cardiomyocyte dysfunction. Injection of LV-piRNA-6426 significantly inhibited the heart dysfunction of rats.piRNA-6426 overexpression inhibits hypoxia-induced cardiomyocyte dysfunction and HF by promoting DNMT3B-mediated methylation of SOAT1 promoter.