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Splicing of pre-mRNAs critically contributes to gene regulation and proteome expansion in eukaryotes, but our understanding of the recognition and pairing of splice sites during spliceosome assembly lacks detail. Here, we identify the multidomain RNA-binding protein FUBP1 as a key splicing factor that binds to a hitherto unknown cis- regulatory motif. By collecting NMR, structural, and in vivo interaction data, we demonstrate that FUBP1 stabilizes U2AF2 and SF1, key components at the 3′ splice site, through multivalent binding interfaces located within its disordered regions. Transcriptional profiling and kinetic modeling reveal that FUBP1 is required for efficient splicing of long introns, which is impaired in cancer patients harboring FUBP1 mutations. Notably, FUBP1 interacts with numerous U1 snRNP-associated proteins, suggesting a unique role for FUBP1 in splice site bridging for long introns. We propose a compelling model for 3′ splice site recognition of long introns, which represent 80% of all human introns. [Display omitted] • FUBP1 recognizes a ubiquitous cis -regulatory RNA motif upstream of the branch point • Multivalent interactions in disordered FUBP1 regions support spliceosome assembly • FUBP1 affects long introns, which are prevalent in humans and altered in cancer • Kinetic modeling and protein interactions implicate FUBP1 in splice site bridging Ebersberger et al. identify the RNA-binding protein FUBP1 as a key splicing factor that binds to a hitherto unknown cis -regulatory motif at 3′ splice sites. Multivalent interactions of FUBP1 with splice site components support spliceosome assembly at multiple stages and ensure efficient splicing of long introns. [ABSTRACT FROM AUTHOR]