Canine mammary tumor (CMT) is one of the relevant models of human breast cancer (HBC) with histopathological, epidemiological, and clinical characteristics similar to those of humans. This study aimed to establish and characterize a new canine cell line CMT-N7. CMT-N7 tumor is a complex canine mammary carcinoma that stained negative for human epidermal growth receptor-2 (HER2) and progesterone receptors (PR), and positive to estrogen receptor (ER). Cell growth, ultrastructure, doubling time, metastasis capacity, and biomarker characteristics of CMT-N7 were assessed. Xenograft transplantation was conducted to evaluate tumorigenicity. The cell morphology of CMT-N7 was generally epithelioid, with large and irregular nuclei and obvious multinucleation. The established CMT-N7 cell line underwent over 120 generations of subculture, exhibiting a rapid proliferation rate with a doubling time of 20.34 h and a chromosome number ranging from 70 to 90. Transwell and wound healing assays demonstrated the CMT-N7 cells had invasive ability. Immunofluorescence analysis revealed positive expression of ER, α-SMA, CK-14, SOX-2, Vimentin, Ki-67, E-cadherin, and COX-2 in CMT-N7 cells. Following inoculation with CMT-N7 cells for two weeks, all mice developed tumors. Immunohistochemical analysis showed negative expression of HER-2 and PR, and positive expression of ER, Ki-67, E-cadherin, Vimentin, and COX-2. Consequently, the establishment of the canine mammary cancer cell line CMT-N7 provides a good model for investigating the mechanism of epithelial-mesenchymal transition (EMT) in both dogs and humans.