Animal shelter workers are a vulnerable population whose exposure to zoonotic disease may be greater compared with the general population. The aim of this project was to identify baseline zoonotic disease knowledge of animal shelter workers and to develop and evaluate zoonotic disease awareness training. Ten animal shelters in six western states were randomly selected. One hundred and eleven trainees were evaluated by identical pre- and post-training tests. Training topics included identification of clinical signs, susceptible species, and transmission of disease to animals and to humans. Zoonotic diseases included rabies, plague, leptospirosis, internal parasites, methicillin-resistant Staphylococcus aureus (MRSA) and salmonella. A statistically significant difference in overall total scores between pre-test (58.5%) and post-test (69.5%) was observed (P = 0.0001). No association was observed between test scores and length of time working in animal shelters, or with the participants’ role at the animal shelter. Overall test scores were raised by 11%. The lowest baseline levels of knowledge were found with leptospirosis, MRSA, plague and rabies, emerging diseases with increasing prevalence and high consequence. Zoonotic disease awareness training is a valuable service to animal shelters. In the current study, training was modestly successful in transferring short-term knowledge to animal shelter workers. To understand and evaluate the effectiveness of training completely, observable or measureable behaviours should be compared before and after training. Long-term assessment with measureable outcomes is needed.