Inflammatory Bowel Disease (IBD) is generally recognised as a dysregulated immune response to bowel flora in genetically susceptible individuals, although its aetiology has never been fully clarified. The similarities with ruminant paratuberculosis, and the isolation of Mycobacterium avium subsp. paratuberculosis(Map) from Crohn's Disease (CD) patient tissues suggest Map as an agent in the pathogenesis of CD. This study was aimed at finding microbiological and immunological evidence of an association between Map and IBD. DNA was extracted from blood of 222 patients and 80 healthy donors from the Basque Country (Spain). Nested PCR for the amplification of Map-specific insertion sequence IS900 was performed, as well as measurement of interferon-γ (IFN-γ ) production in whole blood stimulated with PPA-3 Map antigen (IFNMap) or with Phosphate Buffered Saline (PBS) (IFNPBS) and antibody absorbed ELISA with PPA-3 Map antigen (ABMap). The analyses showed that 17.1% of IBD patients and 42.5% of controls were positive to IS900. IBD patients showed lower IFNMAP production and higher ABMap (specific response) as well as higher IFNPBS production (non-specific response) compared to controls. Statistical analyses showed significant interactions between Map DNAaemia and disease group for IFNPBS and ABMap. Treatment was associated with decreases in IFNMap and PCR-positive frequency. These results suggest the existence of type I and type II immune responses in blood of both healthy controls and IBD patients related to the presence of Map DNA. Pathogenetic models of other mycobacterial infections such as paratuberculosis, tuberculosis and leprosy, where a large fraction of the population is infected but never become clinical, possibly in relationship with a genetic immune dysregulation, support a Map aetiology hypothesis for CD.