<p>Almost all protein-coding and long noncoding genes that are transcribed by RNA polymerase II employ cleavage and polyadenylation (CPA) for 3' end maturation of their nascent RNAs. More than 70% of human mRNA genes display alternative polyadenylation (APA), resulting in expression of isoforms using different CPA sites (also known as poly(A) sites or PAS). APA isoforms often have distinct mRNA metabolism and/or contain variable coding sequences. PAS mutations and genetic variations have been implicated in a growing number of human pathological conditions, underscoring the importance of PAS for proper gene expression. Here we review approaches that modulate the usage of specific PAS and discuss some of their applications in the context of human diseases. We provide our perspectives on current challenges and future directions of strategies of PAS modulation for studying APA isoforms and perturbing gene expression as a therapeutic modality.</p>