Laminitis is regarded as an important underlying cause of lameness disorders, yet its specific pathogenesis remains unclear. Consistent histological changes in cows with laminitis consist in the degradation of the basement membrane (BM). The breakdown of BM is accomplished by numerous proteases, especially MMP-9 and MMP-2. This study recruited 45 cows according to veterinary diagnostic criteria and divided them into three groups: subclinical laminitis cows (SCL, n = 15), chronic laminitis cows (CL, n = 15), and healthy cows (CON, n = 15). After blood samples were collected from the jugular vein, the serum was separated and frozen at –80°C. The serum samples were analyzed by gelatin zymography and reverse zymography to evaluate the activities of MMP-9, MMP-2, TIMP-1, and TIMP-2. In the SCL group, the activity of MMP-9 significantly increased (P <0.01) and the activity of TIMP-1 significantly decreased (P <0.05), compared with those in the CON group, while the activities of MMP-2 and TIMP-2 were not significantly different. In the CL group, the activities of MMP-9 (P <0.001) and MMP-2 (P <0.05) significantly increased, and the activities of TIMP-1 (P <0.01) and TIMP-2 (P <0.05) significantly decreased compared with those in the CON group. This is the first study to report changes in the content of MMP-2 and MMP-9 and their inhibitors, TIMP-2 and TIMP-1, in the serum of dairy cows with laminitis. These results indicate that inadequate regulation of the activities of MMPs and TIMPs in serum may play a role in the development of laminitis in dairy cows.