Mycobacterium avium subsp. paratuberculosis is a zoonotic pathogen whose association with Crohn's disease in humans is under scrutiny. The objective of this work was to investigate its association with other chronic diseases such as type 1 diabetes mellitus (T1DM), where the involvement of a persistent pathogen such as M. avium subsp. paratuberculosis could be the trigger. For this purpose, 59 diabetic patients and 59 healthy controls were investigated for the presence of antibodies against two recombinant proteins of M. avium subsp. paratuberculosis and the whole-cell lysate. Extremely significant humoral immune responses to recombinant heparin binding hemagglutinin and glycosyl transferase proteins and the whole-cell lysates of M. avium subsp. paratuberculosis bacilli were observed in T1DM patients and compared to those of healthy controls. Finding evidence of M. avium subsp. paratuberculosis involvement in T1DM is perhaps a novel finding that might serve as a foundation stone in establishing an infectious etiology for T1DM.