AbstractThe polyA tail of mRNAs is important for many aspects of RNA metabolism. However, whether and how it regulates pre‐mRNA splicing is still unknown. Here, we report that the polyA tail acts as a splicing enhancer for the last intron via the nuclear polyA binding protein PABPN1 in HeLa cells. PABPN1‐depletion induces the retention of a group of introns with a weaker 3′ splice site, and they show a strong 3′‐end bias and mainly locate in nuclear speckles. The polyA tail is essential for PABPN1‐enhanced last intron splicing and functions in a length‐dependent manner. Tethering PABPN1 to nonpolyadenylated transcripts also promotes splicing, suggesting a direct role for PABPN1 in splicing regulation. Using TurboID‐MS, we construct the PABPN1 interactome, including many spliceosomal and RNA‐binding proteins. Specifically, PABPN1 can recruit RBM26&27 to promote splicing by interacting with the coiled‐coil and RRM domain of RBM27. PABPN1‐regulated terminal intron splicing is conserved in mice. Together, our study establishes a novel mode of post‐transcriptional splicing regulation via the polyA tail and PABPN1.