Aberrant patterns of pre-mRNA processing are typical of human malignancies, yet the mechanisms responsible for these changes remain undefined. We have recently shown overexpression of a core splice regulatory protein, serine-arginine protein kinase 1 (SRPK1), in dysplastic and neoplastic pancreatic ductular cells. In the present study, we have established that SRPK1 levels are similarly up-regulated in breast and colonic tumors where its expression increases coordinately with tumor grade. Targeting SRPK1 for inhibition using small interfering RNA in breast and colonic tumor cell lines in vitro resulted in both increased apoptotic potential and enhanced cell killing after treatment with gemcitabine and cisplatin. Recent reports have described multifaceted interactions between the mitogen-activated protein kinase (MAPK) and AKT signaling networks and the splice regulatory machinery. Consequently, we have shown that targeted inhibition of SRPK1 in tumor cells results in reduced phosphorylation of MAPK3, MAPK1, and AKT. Alterations in the splice pattern and resulting expression of MAPK kinase are implicated in mediating the antitumoral effects resulting from SRPK1 down-regulation. The up-regulation of SRPK1 in multiple cancers and its ability to regulate multiple relevant signaling pathways provide support for developing agents to inhibit this kinase for possible broad application to treat epithelial cancers. [Cancer Res 2007;67(5):2072–80]