We investigated the gene expression of matrix metalloproteinases-9 (MMP-9) and tissue inhibitors of matrix metalloproteinases-1 (TIMP-1)in peripheral blood cells from infected cattle with Mycobacterium avium subsp. paratuberculosis (Map) in the ELISA-negative subclinical stage compared with uninfected control cattle. MMP-9 and TIMP-1 gene expression analysis was carried out using real-time RT-PCR. The activity of MMP-9 was analyzed by gelatin zymography. Significant decreased MMP-9 expression andincreased TIMP-1 expression were found in peripheral blood cells from Map-infected cattle after stimulation with Map lysate and Map purified protein derivative (PPD) than in controlcattle by real-time RT-PCR analysis. In contrast to the uninfected controls, the activity of MMP-9 was also decreased in peripheralblood cell culture supernatants from Map-infected cattle at 24h after Map lysate and Map PPD stimulation by gelatin zymography analysis. So the MMP-9 may play an important role in the development of Mycobacterium avium subsp. paratuberculosis disease.