Splicing and 3′‐end processing (including cleavage and polyadenylation) of vertebrate pre‐mRNAs are tightly coupled events that contribute to the extensive molecular network that coordinates gene expression. Sequences within the terminal intron of genes are essential to stimulate pre‐mRNA 3′‐end processing, although the factors mediating this effect are unknown. Here, we show that the pyrimidine tract of the last splice acceptor site of the human β‐globin gene is necessary to stimulate mRNA 3′‐end formation in vivo and binds the U2AF 65 splicing factor. Naturally occurring β‐thalassaemia‐causing mutations within the pyrimidine tract reduces both U2AF 65 binding and 3′‐end cleavage efficiency. Significantly, a fusion protein containing U2AF 65, when tethered upstream of a cleavage/polyadenylation site, increases 3′‐end cleavage efficiency in vitro and in vivo. Therefore, we propose that U2AF 65 promotes 3′‐end processing, which contributes to 3′‐terminal exon definition.