Canine mammary tumors (CMTs) have been proposed to be a good animal model for human breast cancer. To provide a basis for the tumorigenic study of CMTs, cell lines were established using a modified cell culture technique. The epithelial morphology and immunostaining with cytokeratin 18 confirmed the epithelial origin of the cells. In an investigation of possible mammary tumorigenesis–related factors, the expression of Wnt signaling–related proteins was detected in cell lines. Secreted frizzled-related protein 2 (SFRP2) was abundantly expressed in CMT cells but not in normal canine mammary gland (MG) cells. Secreted frizzled-related protein 2 was secreted into the culture medium and was associated with the extracellular matrix. In addition, increased expressions of β-catenin and cyclin D1 were observed in cells overexpressing SFRP2. The marked differential expression of SFRP2 reveals that this protein may be a potential candidate marker for CMTs. The CMT cell line established in this study provides a useful tool and experimental model for understanding both the tumorigenesis of CMTs and the role of Wnt signaling in cancers.