Antiproliferative factor (APF), a Frizzled-8 protein-related sialoglycopeptide involved in the pathogenesis of interstitial cystitis, potently inhibits proliferation of normal urothelial cells as well as certain cancer cells. To elucidate the molecular mechanisms of the growth-inhibitory effect of APF, we performed stable isotope labeling by amino acids in cell culture analysis of T24 bladder cancer cells treated with and without APF. Among over 2000 proteins identified, 54 were significantly up-regulated and 48 were down-regulated by APF treatment. Bioinformatic analysis revealed that a protein network involved in cell adhesion was substantially altered by APF and that β-catenin was a prominent node in this network. Functional assays demonstrated that APF down-regulated β-catenin, at least in part, via proteasomal and lysosomal degradation. Moreover, silencing of β-catenin mimicked the antiproliferative effect of APF whereas ectopic expression of nondegradable β-catenin rescued growth inhibition in response to APF, confirming that β-catenin is a key mediator of APF signaling. Notably, the key role of β-catenin in APF signaling is not restricted to T24 cells, but was also observed in an hTERT-immortalized human bladder epithelial cell line, TRT-HU1. In addition, the network model suggested that β-catenin is linked to cyclooxygenase-2 (COX-2), implying a potential connection between APF and inflammation. Functional assays verified that APF increased the production of prostaglandin E2 and that down-modulation of β-catenin elevated COX-2 expression, whereas forced expression of nondegradable β-catenin inhibited APF-induced up-regulation of COX-2. Furthermore, we confirmed that β-catenin was down-regulated whereas COX-2 was up-regulated in epithelial cells explanted from IC bladder biopsies compared with control tissues. In summary, our quantitative proteomics study describes the first provisional APF-regulated protein network, within which β-catenin is a key node, and provides new insight that targeting the β-catenin signaling pathway may be a rational approach toward treating interstitial cystitis.