MicroRNAs (miRNAs) are thought to act as post-transcriptional regulators in the cytoplasm by either dampening translation or stimulating degradation of target mRNAs. With the increasing resolution and scope of RNA mapping, recent studies have revealed novel insights into the subcellular localization of miRNAs. Based on miRNA subcellular localization, unconventional functions and mechanisms at the transcriptional and post-transcriptional levels have been identified. This minireview provides an overview of the subcellular localization of miRNAs and the mechanisms by which they regulate transcription and cellular homeostasis in mammals, with a particular focus on the roles of phase-separated biomolecular condensates.