CUGBP2 (ETR-3/NAPOR/BRUNOL3) promotes inclusion of cardiac troponin T (cTNT) exon 5 via binding between positions 21 and 74 of the downstream intron. The molecular mechanism by which CUGBP2 activates cTNT exon 5 inclusion is unknown. Our results suggest that CUGBP2 promotes exon inclusion by a novel mechanism in which CUGBP2 directly interacts with components of the activated U2 snRNP and enhances binding of U2 snRNP to the branch site located upstream of the exon. Using an in vitro splicing assay, we show that recombinant CUGBP2 enhances complex A formation of a cTNT pre-mRNA. Enhanced complex A assembly requires both the upstream and downstream introns consistent with dual requirements for the downstream CUGBP2-binding site and an upstream branch site for U2 snRNP binding. We also show that CUGBP2 enhances binding of U2 snRNA to the cTNT pre-mRNA consistent with enhanced complex A assembly. Purification of CUGBP2-interacting proteins using tandem affinity purification leads to the demonstration that the core 17S U2 snRNP components, SF3b145 and SF3b49 bind directly to CUGBP2. We conclude that CUGBP2 activates exon inclusion by forming direct interactions with components of the 17S snRNP complex and recruits and/or stabilizes binding of U2 snRNP.