For the 2003 monkeypox virus (MPXV) outbreak in the United States, interhuman transmission was not documented and all case-patients were near or handled MPXV-infected prairie dogs. We initiated a case-control study to evaluate risk factors for animal-to-human MPXV transmission. Participants completed a questionnaire requesting exposure, clinical, and demographic information. Serum samples were obtained for analysis of immunoglobulin G (IgG) and IgM to orthopoxvirus. When data were adjusted for smallpox vaccination, case-patients were more likely than controls to have had daily exposure to a sick animal (odds ratio [OR] 4.0, 95% confidence interval [CI] 1.2-13.4), cleaned cages and bedding of a sick animal (OR 5.3, 95% CI 1.4-20.7), or touched a sick animal (OR 4.0, 95% CI 1.2-13.4). These findings demonstrate that human MPXV infection is associated with handling of MPXV-infected animals and suggest that exposure to excretions and secretions of infected animals can result in infection.