Objective: To evaluate the in vitro susceptibility of various field isolates of Mycobacterium avium subsp paratuberculosis (MAP) to gallium nitrate. Sample: 10 isolates of MAP, including 4 isolated from cattle, 2 isolated from bison, 1 isolated from an alpaca, and 3 isolated from humans. Procedures: The in vitro susceptibility to gallium nitrate was tested by use of broth culture with detection of MAP growth by means of a nonradiometric automated detection method. For each MAP isolate, a series of 7 dilutions of gallium nitrate (concentrations ranging from 200 to 1,000μM) were tested. Gallium nitrate was considered to have caused 90% and 99% inhibition of the MAP growth when the time to detection for culture of the MAP stock solution and a specific concentration of gallium nitrate was delayed and was similar to that obtained for culture of the MAP stock solution (without the addition of gallium nitrate) diluted 1:10 and 1:100, respectively. Results: Gallium nitrate inhibited MAP growth in all 10 isolates. The susceptibility to gallium nitrate was variable among isolates, and all isolates of MAP were inhibited in a dose-dependent manner. Overall, the concentration that resulted in 90% inhibition ranged from < 200μM for the most susceptible isolates to 743μM for the least susceptible isolates. Conclusions and Clinical Relevance: Gallium nitrate had activity against all 10 isolates of MAP tested in vitro and could potentially be used as a prophylactic agent to aid in the control of MAP infections during the neonatal period.