RBM25 has been shown to associate with splicing cofactors SRm160/300 and assembled splicing complexes, but little is known about its splicing regulation. Here, we characterize the functional role of RBM25 in alternative pre-mRNA splicing. Increased RBM25 expression correlated with increased apoptosis and specifically affected the expression of Bcl-x isoforms. RBM25 stimulated proapoptotic Bcl-x(S) 5' splice site (5' ss) selection in a dose-dependent manner, whereas its depletion caused the accumulation of antiapoptotic Bcl-x(L). Furthermore, RBM25 specifically bound to Bcl-x RNA through a CGGGCA sequence located within exon 2. Mutation in this element abolished the ability of RBM25 to enhance Bcl-x(S) 5' ss selection, leading to decreased Bcl-x(S) isoform expression. Binding of RBM25 was shown to promote the recruitment of the U1 small nuclear ribonucleoprotein particle (snRNP) to the weak 5' ss; however, it was not required when a strong consensus 5' ss was present. In support of a role for RBM25 in modulating the selection of a 5' ss, we demonstrated that RBM25 associated selectively with the human homolog of yeast U1 snRNP-associated factor hLuc7A. These data suggest a novel mode for Bcl-x(S) 5' ss activation in which binding of RBM25 with exonic element CGGGCA may stabilize the pre-mRNA-U1 snRNP through interactions with hLuc7A.