AbstractPolyadenylation of pre-messenger RNA (pre-mRNA) specific sites and termination of their downstream transcriptions are signaled by unique sequence motif structures such as AAUAAA and its auxiliary elements. Alternative polyadenylation (APA) is an important post-transcriptional regulatory mechanism that processes RNA products depending on its 3′-untranslated region (3′-UTR) specific sequence signal. APA processing can generate several mRNA isoforms from a single gene, which may have different biological functions on their target gene. As a result, cellular genomic stability, proliferation capability, and transformation feasibility could all be affected. Furthermore, APA modulation regulates disease initiation and progression. APA status could potentially act as a biomarker for disease diagnosis, severity stratification, and prognosis forecast. While the advance of modern throughout technologies, such as next generation-sequencing (NGS) and single-cell sequencing techniques, have enriched our knowledge about APA, much of APA biological process is unknown and pending for further investigation. Herein, we review the current knowledge on APA and how its regulatory complex factors (CFI/IIm, CPSF, CSTF, and RBPs) work together to determine RNA splicing location, cell cycle velocity, microRNA processing, and oncogenesis regulation. We also discuss various APA experiment strategies and the future direction of APA research.