It is known that epithelial mesenchymal transition (EMT) contributes to the acquisition of malignant property in human cancers. However, the role of EMT in canine tumors remains to be elucidated. To evaluate the correlation between expression levels of protein markers involved in EMT and clinicopathological characteristics in canine mammary gland tumors, immunohistochemistry using antibodies against ZO-1, E-cadherin, vimentin, N-cadherin and fibronectin was performed on 119 clinical tissue samples. Consequently, loss of ZO-1 and E-cadherin, and gain of vimentin and N-cadherin were more frequently observed in malignant tumors than in benign tumors. However, there was no correlation among expression of these molecules. Univariate and multivariate analysis identified that loss of E-cadherin independently had a low one-year survival rate (adjusted odds ratio: 2.3, P=0.02). These results suggested that EMT might relate to acquisition of malignancy, and additionally, E-cadherin was strongly correlated with malignant behavior in canine mammary gland tumors.