In 2009, the Tennessee Department of Health received reports of 5 tuberculin skin test (TST) conversions among employees of an elephant refuge and isolation of Mycobacterium tuberculosis from a resident elephant. To determine the extent of the outbreak and identify risk factors for TST conversion, we conducted a cohort study and onsite assessment. Risk for conversion was increased for elephant caregivers and administrative employees working in the barn housing the M. tuberculosis-infected elephant or in offices connected to the barn (risk ratio 20.3, 95% confidence interval 2.8-146.7). Indirect exposure to aerosolized M. tuberculosis and delayed or inadequate infection control practices likely contributed to transmission. The following factors are needed to reduce risk for M. tuberculosis transmission in the captive elephant industry: increased knowledge about M. tuberculosis infection in elephants, improved infection control practices, and specific occupational health programs.