OBJECTIVE To identify knowledge and practices related to rabies vaccination and serologic monitoring among animal care workers in the United States. DESIGN Cross-sectional survey. SAMPLE 2,334 animal care workers (ie, veterinarians, veterinary technicians, animal control workers, and wildlife rehabilitators). PROCEDURES Participants were contacted through relevant professional organizations to participate in an anonymous web-based survey. The survey collected demographic and occupational information, animal handling and potential rabies exposure information, and individual rabies vaccination and serologic monitoring practices. Comparisons of animal bite and rabies exposure rates were made between occupational groups. Multiple logistic regression was used to evaluate factors associated with rabies vaccination status and adherence to serologic monitoring recommendations. RESULTS Respondents reported 0.77 animal bites/person-year or 0.10 bites/1,000 animals handled. The overall rate of postexposure prophylaxis due to an occupational rabies exposure was 1.07/100 person-years. Veterinarians reported the highest rabies vaccination rate (98.7% [367/372]), followed by animal control workers (78.5% [344/438]), wildlife rehabilitators (78.2% [122/156]), and veterinary technicians (69.3% [937/1,352]). Respondents working for employers requiring rabies vaccination and serologic monitoring were 32.16 and 6.14 times, respectively, as likely to be vaccinated or have a current serologic monitoring status as were respondents working for employers without such policies. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that, given the high reported rates of animal bites and potential rabies exposures among animal care workers, improvements in rabies vaccination and serologic monitoring practices are needed.