Since the discovery of klotho as an anti-aging gene, its association with tumors has been studied. Several previous studies have reported the down-expression of klotho in various human cancers, and much of its mechanism has been revealed. Nonetheless, the significance of klotho in canine mammary gland tumors is not yet known. This study aimed to determine whether klotho is expressed within normal canine mammary glands and whether the expression changes in benign and malignant tumors. Using immunohistochemistry, the experiment was conducted on eight normal canine mammary gland tissues and 55 mammary gland tumor samples. Additionally, the correlation between the Ki-67 proliferation index and clinicopathological features, such as age, tumor size, tumor grade, histologic type, and metastasis, was evaluated. All eight normal mammary gland tissues showed immunohistochemistry expression of klotho, and the expression significantly decreased as malignancy increased. Among the samples, 11% (3/28) of benign tumors and 26% (7/27) of malignant tumors showed negative klotho expression. Furthermore, higher Ki-67 expression, higher grades, and metastasis were confirmed to be associated with the negative klotho expression. Analysis of the survival curve for dogs with malignant tumors revealed that negative klotho expression was significantly associated with poor overall survival and disease-free survival. These results indicate that klotho is expressed in normal canine mammary glands and that negative klotho expression in canine mammary gland tumors is positively correlated with poor prognosis.