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 beta-globin gene is necessary to stimulate mRNA 3'-end formation in vivo and binds the U2AF 65 splicing factor. Naturally occurring beta-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.