Selenium (Se) is imperative for normal myocardial differentiation and development, and these basic cellular functions can be regulated by miRNA during cardiogenesis. Here, we show that Se deficiency can cause defects in myocardial development and abnormalities of cardiomyocyte differentiation. In previous work using microRNAome analysis, we found that miR-215-5p was differentially expressed in Se-deficient myocardial tissues. However, the relationship between miR-215-5p and Se deficiency in myocardial development remains unknown. In this study, CCCTC-binding factor (CTCF) was confirmed as the target gene of miR-215-5p by dual luciferase reporter assay, western blot and quantitative real-time polymerase chain reaction (qRT-PCR) in cardiomyocytes. Based on in vivo and in vitro results, we found that the increased expression of miR-215-5p induced by Se deficiency may cause transcriptional disorders of myocardial genes, mitochondrial biosynthesis imbalance, and a reduction of myocardial development and differentiation-related factors. Moreover, miR-215-5p may target CTCF to regulate myocardial development and differentiation via the noncanonical Wnt signaling pathway and induce mitochondrial dysfunction via the PGC-1α-TFAM-NRF1/2 pathway in the heart. Our results not only demonstrated that Se deficiency affected myocardial development and differentiation by directly targeting the miR-215-5p/CTCF axis but also found that miR-215-5p inhibitor promoted normal differentiation of cardiomyocytes and myocardial development and ameliorated myocardium structural abnormalities via the noncanonical Wnt signaling pathway in chicken. Our findings support the potential of applying miRNAs during the process of cardiogenesis and indicate that miR-215-5p could be a novel candidate for treatment of cardiac hypoevolutism.