The human CD44 gene undergoes extensive alternative splicing of multiple variable exons positioned in a cassette in the middle of the gene. Expression of alternative exons is often restricted to certain tissues and could be associated with tumor progression and metastasis of several human malignancies, including breast cancer. Exon v4 contains multiple copies of a C/A-rich exon enhancer sequence required for optimal inclusion of the exon and binding to the nucleic acid-binding proteins YB-1 and human Tra2-beta1. Here, we show that hTra2-beta1, a member of the extended family of serine/arginine-rich (SR) splicing factors, enhances the in vivo inclusion of CD44 exons v4 and v5. It increased inclusion of exons v4 and v5 and acted synergistically with YB-1. Activation required the C/A-rich enhancer within exon v4. Several other SR proteins had none or only a slight effect on CD44 exon inclusion. In contrast, SC35 inhibited exon usage and antagonized the effects of Tra2 or YB-1. In a matched pair analysis of human breast cancers and their corresponding nonpathologic tissue controls, we found a significant induction of Tra2-beta1 in invasive breast cancer, both on the RNA and protein levels. Together with our functional data, these results suggest an important role for Tra2-beta1 in breast cancer. Induction of this splicing factor might be responsible for splicing of CD44 isoforms associated with tumor progression and metastasis.