Osteopontin (Opn), a highly acidic glycoprotein, promotes cellular adhesion and recruitment and has been shown to be upregulated in the granulomas of mycobacterial infections. Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is associated with granulomatous enteritis. The objective of this experiment was to identify Opn in the ileum and ileocecal lymph node (ICN) of dairy cows naturally infected with MAP and to compare the frequency and intensity of staining between noninfected healthy controls, subclinical and clinical cows. Sections from these three groups of animals were selected from a tissue archive. Immunohistochemical analysis was used to determine the location and expression of Opn. The frequency and intensity of staining was also reported. Confirmation of acid-fast bacilli in the tissue sections was achieved by the Ziehl-Neelsen method. Within the ileal tissue, macrophages, lymphocytes, and plasma cells stained positive for Opn. Clinical cows expressed Opn at a greater frequency in the lamina propria. Control and subclinical cows did not have areas of granulomatous inflammation but cells staining for Opn were equally intense for the three groups. The frequency of staining for Opn in the ICN was not affected by MAP infection. Results of this study confirm for the first time, the expression of Opn in the ileum and ICN of MAP-infected cattle.