Serine protease PRSS23 is a newly discovered protein that has been associated with tumor progression in various types of cancers. Interestingly, PRSS23 is coexpressed with estrogen receptor alpha (ERalpha), which is a prominent biomarker and therapeutic target for human breast cancer. Estrogen signaling through ERalpha is also known to affect cell proliferation, apoptosis, and survival, which promotes tumorigenesis by regulating the production of numerous downstream effector proteins.In the present study, we aimed to clarify the correlation between and functional implication of ERalpha and PRSS23 in breast cancer. Analysis of published breast cancer microarray datasets revealed that the gene expression correlation between ERalpha and PRSS23 is highly significant among all ERalpha-associated proteases in breast cancer. We then assessed PRSS23 expression in 56 primary breast cancer biopsies and 8 cancer cell lines. The results further confirmed the coexpression of PRSS23 and ERalpha and provided clinicopathological significance. In vitro assays in MCF-7 breast cancer cells demonstrated that PRSS23 expression is induced by 17beta-estradiol-activated ERalpha through an interaction with an upstream promoter region of PRSS23 gene. In addition, PRSS23 knockdown may suppress estrogen-driven cell proliferation of MCF-7 cells.Our findings imply that PRSS23 might be a critical component of estrogen-mediated cell proliferation of ERalpha-positive breast cancer cells. In conclusion, the present study highlights the potential for PRSS23 to be a novel therapeutic target in breast cancer research.