Vaccinia virus (VACV) is used commonly in research laboratories. Non-highly attenuated strains of VACV are potentially pathogenic in humans, and VACV vaccination and biosafety level 2 facilities and protocols are currently recommended for vaccinated laboratory workers in the United States who handle non-highly attenuated strains of the virus. Despite this, laboratory-related VACV exposures continue to occur and a number of recent instances of VACV infection in non-vaccinated laboratory workers have been documented. We provide a discussion of the usage and risks associated with VACV in laboratory research.