This Special Issue delves into the intricate world of miRNAs as modulators of gene expression regulation. These small non-coding RNAs are at the heart of post-transcriptional modulation of gene activity, affecting a myriad of biological processes. The curated selection of articles within this issue offers insights into the roles of miRNAs in health and disease, emphasizing their potential value as therapeutic targets and biomarkers. Featuring original research and in depth-reviews, this Special Issue covers recent miRNA advancements, detailing their regulatory networks and action mechanisms. It highlights new miRNA-linked therapeutic targets and circulating miRNAs as cell-to-cell communication intermediaries. The issue also examines extracellular miRNAs (ex-miRNAs) and their functions, highlighting their presence in biological fluids and their potential utility in clinical diagnostics and disease monitoring. Challenges in miRNA quantification and technological advances for precise measurement are also discussed. The issue traverses the less-charted territories of miRNA research, such as the discovery of small nucleolar-derived miRNAs and the elucidation of transposable elements as unexpected constituents of the miRNA regulatory network. Overall, this collection showcases the evolving nature of miRNA research, offering valuable perspectives for future scientific inquiry and medical innovation. It reflects the drive to decode gene regulation complexities, providing insights for health professionals and researchers at the forefront of gene regulation research.