We describe animal-to-human transmission of SARS-CoV-2 in a zoo setting in Indiana, USA. A vaccinated African lion with physical limitations requiring hand feeding tested positive for SARS-CoV-2 after onset of respiratory signs. Zoo employees were screened, monitored prospectively for onset of symptoms, then rescreened as indicated; results were confirmed by using reverse transcription PCR and whole-genome virus sequencing when possible. Traceback investigation narrowed the source of infection to 1 of 6 persons. Three exposed employees subsequently had onset of symptoms, 2 with viral genomes identical to the lion’s. Forward contact tracing investigation confirmed probable lion-to-human transmission. Close contact with large cats is a risk factor for bidirectional zoonotic SARS-CoV-2 transmission that should be considered when occupational health and biosecurity practices at zoos are designed and implemented. SARS-CoV-2 rapid testing and detection methods for big cats and other susceptible animals should be developed and validated to enable timely implementation of One Health investigations.