Whether Mycobacterium ulcerans, the etiological agent of Buruli ulcer in numerous tropical countries, would exist in a dormant state as reported for closely related Mycobacterium species, has not been established. Six M. ulcerans strains were exposed to a progressive depletion in oxygen for 2 months, using the Wayne model of dormancy previously described for M. tuberculosis, and further examined by microscopy after staining of dynamic, dormant, and dead mycobacteria (DDD staining), microcalorimetry and subculture in the presence of dead and replicative M. ulcerans as controls. Mycobacterium ulcerans CU001 strain died during the progressive oxygen depletion and four of five remaining strains exhibited Nile red-stained intracellular lipid droplets and a 14- to 20-day regrowth when exposed to ambient air, consistent with dormancy. A fifth M. ulcerans 19423 strain stained negative in DDD staining and slowly regrew in 27 days. Three tested M. ulcerans strains yielded microcalorimetric pattern similar to that of the negative (dead) homologous controls, differing from that of the homologous positive (replicative) controls. The relevance of these experimental observations, suggesting a previously unreported dormancy state of M. ulcerans, warrants further investigations in the natural ecological niches where M. ulcerans thrive as well as in Buruli ulcer lesions.