In order to understand the change of MMP-2 and MMP-9 in dogs with mammary gland tumor (MGT), the activity of MMP-2 and MMP-9 in plasma samples, MGT tissues and near by normal mammary gland tissues were evaluated by gelatin zymography. The levels of MMP-2 and MMP-9 were significantly higher in tumor tissues than those in normal gland tissues (p<0.05). Plasma MMP-9 levels in malignant cases were higher than those in benign (p=0.15) and non-tumor cases (p<0.05). Likewise, plasma MMP-2 levels in malignant MGT cases were higher than non-tumor (p<0.01) and benign cases (p=0.54)). Furthermore, plasma MMP-9 levels were reflected and positively correlated with MMP-9 in tumor tissues (r=0.78, p=0.02), but not MMP-2. Our results indicated that the MMP-2 and MMP-9 levels are associated with tumor malignancy, and plasma MMP-9 activity can become a potential marker both in follow-up and prognosis of canine MGT patients.