In recent years, systematic analyses of the subcellular distribution of microRNAs (miRNAs) suggest that the majority of miRNAs are present in both nuclear and cytoplasmic compartments. However, the full extent of nuclear miRNA function in cardiomyocytes is currently unknown. Here, subcellular fractionation, followed by the miRNA microarray, revealed that most miRNAs were detectable in both nuclear and cytoplasmic fractions of cardiomyocytes. We employed miR-320 as an example to explore the function of nucleus-localized miRNAs, finding that CRISPR-Cas9-mediated Ago2 knockdown abolished miR-320-induced transcriptional remodeling. Furthermore, nuclear Ago2 re-expression restored the effects of miR-320 in the nucleus. Moreover, liquid chromatographymass spectrometry (LC-MS) analysis revealed the association of nuclear Ago2 with transcription factors YLP motif-containing protein 1 (Ylpm1) and single-stranded DNA binding protein 1 (Ssbp 1 ). Intersection of the data of transcriptome-sequencing (seq) with Ago2-chromatin immunoprecipitation (ChIP)-seq revealed that the binding of Ago2 with the target promoter DNA may require promoter RNAs. Specifically, Cep57 was upregulated, whereas Fscn2 was downregulated by miR-320, and a similar effect was also observed by knockdown of their promoter RNA, respectively. Chromatin isolation by RNA purification (ChIRP) analysis showed decreased binding of the Cep57 and Fscn2 promoter RNA on their promoter DNA by miR-320 overexpression.Our work provided a preliminary idea that promoter RNA transcripts act as "pioneers" to disrupt chromatin that permits Ago2/miR-320 complexes to target Cep57 or Fscn2 promoter DNA for transcriptional regulation. miRNAs are naturally located in both cytoplasm and nucleus; however, their pathophysiological functions are largely unknown. Our work provided a theoretical basis for developing nuclear miRNA-based therapeutics against various diseases in the future.