Two co-housed 17-yr-old male lions (Panthera leo) and one 15-yr-old female lion in an adjacent enclosure developed acute lethargy, depression, anorexia, and ataxia at a zoological park in central North Carolina, United States. One of the male lions and the female lion were found dead 5 and 4 d after the onset of clinical signs, respectively. The other male lion recovered without any clinical treatment. A third male lion housed with the female lion never developed clinical signs. Postmortem examination, microscopic evaluation, and bacterial culture of the liver from both deceased lions confirmed systemic Yersinia pseudotuberculosis infection. Susceptibility testing revealed resistance to amoxicillin-clavulanic acid and cefazolin. To investigate the extent of the outbreak, qualitative real-time polymerase chain reaction (PCR) for Y. pseudotuberculosis was performed on feces and substrate from 15 enclosures housing nondomestic felids and canids, resulting in a positive sample from one enclosure housing four asymptomatic lions. This enclosure was adjacent to housing of the deceased female lion. Enrofloxacin was administered to all animals in the vicinity at doses of 5 mg/kg in nondomestic felids and 10 mg/kg in nondomestic canids, orally q24h for 14 d. Repeated fecal PCR performed 1 wk after completing the antibiotic treatment protocol found no positive samples. The source of the infection was not identified despite PCR testing of environmental samples from all enclosures in the vicinity, the remains of a prey item fed out prior to the outbreak, and a single dead rodent found on grounds. No further clinical cases have occurred within the following year.