Background Prostatitis is associated with a characteristic increase in prostatic fluid pH; however, the underlying mechanism and its physiological significance have not been elucidated. Methodology/Principal Findings In this study a primary culture of rat prostatic epithelial cells and a rat prostatitis model were used. Here we reported the involvement of CFTR, a cAMP-activated anion channel conducting both Cl− and HCO3−, in mediating prostate HCO3− secretion and its possible role in bacterial killing. Upon Escherichia coli (E coli)-LPS challenge, the expression of CFTR and carbonic anhydrase II (CA II), along with several pro-inflammatory cytokines was up-regulated in the primary culture of rat prostate epithelial cells. Inhibiting CFTR function in vitro or in vivo resulted in reduced bacterial killing by prostate epithelial cells or the prostate. High HCO3− content (>50 mM), rather than alkaline pH, was found to be responsible for bacterial killing. The direct action of HCO3− on bacterial killing was confirmed by its ability to increase cAMP production and suppress bacterial initiation factors in E coli. The relevance of the CFTR-mediated HCO3− secretion in humans was demonstrated by the upregulated expression of CFTR and CAII in human prostatitis tissues. Conclusions/Significance The CFTR and its mediated HCO3− secretion may be up-regulated in prostatitis as a host defense mechanism.