In a deer farm, chronic diarrhoea was seen in a 4-year-old hind. This animal died in poor condition on the farm and Johne's disease was suspected. Ziehl-Neelsen staining of the faeces of this hind were positive for the presence of clumps of small acid-fast bacilli, but faecal cultures remained negative. Direct and indirect tests were performed on 24 hinds and stags (yearlings, 2- and 4-year-old animals). The indirect tests performed were serology (Mycobacterium paratuberculosis antibody ELISA, HerdChek, Idexx), comparative cervical skin test (CCT) and lymphoproliferation test (LT) using Mycobacterium bovis purified protein derivative (PPD) and Mycobacterium avium PPD as antigens. Three positive serological results, three positive CCT and eight positive LT were observed in hinds and stags older than 2 years. No positive serological results were observed in the yearling group, whereas some sensitisation was observed in the CCT as well as in the LT for the same group of animals. The degree of concordance between these indirect tests was poor. The three seropositive animals were slaughtered and subjected to post-mortem examination. Histopathology was performed on mesenteric lymph nodes and on the terminal ileum. Visual changes in some mesenteric lymph nodes were observed, no gross lesion was seen in the intestine. Although Ziehl-Neelsen staining yielded no positive results, a catarrhal focal necrotic enteritis associated with a granulomatous lymphadenitis compatible with Johne's disease was evidenced. The mycobacterial cultures on organ samples from slaughtered animals were positive after 2 months for M. avium subspecies paratuberculosis and negative for M. bovis and M. avium. This is the first description of Johne's disease in a deer farm in Belgium.