Alternative polyadenylation (APA) is a widespread RNA-processing mechanism that generates distinct 3′ termini on mRNAs and other RNA polymerase II transcripts. It plays a crucial role in gene regulation, tissue specificity, and cell proliferation and differentiation. APA can occur in both 3′ untranslated regions (3′ UTRs) and upstream regions (URs) of genes, leading to the production of mRNA isoforms with varying lengths of 3′ UTRs or terminal exons. These isoforms can affect mRNA stability, translation, nuclear export, and protein localization, thereby influencing gene expression and cellular functions. The molecular mechanisms underlying APA involve variations in the concentration of core processing factors and RNA-binding proteins, as well as transcription-based regulation. Core polyadenylation factors, such as CSTF64 and PABPN1, play significant roles in regulating APA, and their expression levels can globally affect 3′ UTR length. Additionally, splicing and transcriptional processes are interconnected with APA, with U1 snRNP and transcription elongation factors influencing PAS choice. The clinical implications of APA are also discussed, highlighting its potential as a therapeutic target and its role in human diseases.Alternative polyadenylation (APA) is a widespread RNA-processing mechanism that generates distinct 3′ termini on mRNAs and other RNA polymerase II transcripts. It plays a crucial role in gene regulation, tissue specificity, and cell proliferation and differentiation. APA can occur in both 3′ untranslated regions (3′ UTRs) and upstream regions (URs) of genes, leading to the production of mRNA isoforms with varying lengths of 3′ UTRs or terminal exons. These isoforms can affect mRNA stability, translation, nuclear export, and protein localization, thereby influencing gene expression and cellular functions. The molecular mechanisms underlying APA involve variations in the concentration of core processing factors and RNA-binding proteins, as well as transcription-based regulation. Core polyadenylation factors, such as CSTF64 and PABPN1, play significant roles in regulating APA, and their expression levels can globally affect 3′ UTR length. Additionally, splicing and transcriptional processes are interconnected with APA, with U1 snRNP and transcription elongation factors influencing PAS choice. The clinical implications of APA are also discussed, highlighting its potential as a therapeutic target and its role in human diseases.