Notch3 gene amplification has recently been identified in ovarian cancer but the Notch3 effectors that are involved in the development of ovarian cancer remain elusive. In this study, we have identified Pbx1, a proto-oncogene in hematopoietic malignancy, as a Notch3 target gene. Pbx1 expression is transcriptionally regulated by Notch3 activation, and Notch3/CSL protein complex directly binds to the Pbx1 promoter segment harboring the CSL-binding sequence. The growth-inhibitory effect of gamma-secretase inhibitor could be partially reversed by ectopic Pbx1 expression. Furthermore, functional studies by Pbx1 short hairpin RNA knockdown show that Pbx1 is essential for cell proliferation and tumorigenicity. Taken together, the above findings indicate that Pbx1 is a direct Notch3-regulated gene that mediates the survival signal of Notch3 in ovarian cancer.