HIV-related criminal laws in some jurisdictions may hamper population health efforts to manage HIV and bring about an AIDS-free generation. HIV care nurses have an instrumental role to play in ensuring equitable care and health for all in a context of HIV. The purpose of our study was to determine HIV care nurses' knowledge of HIV-related criminal laws. Ecosocial theory and content expert opinion guided development of a questionnaire to assess nurses' knowledge of HIV-related criminal laws. A total of 174 HIV care nurses from Canada (n = 23) and the United States (n = 151) completed the questionnaire. Knowledge gaps were observed in several aspects of HIV-related criminal laws that can influence nursing clinical practices. Nurses should increase their knowledge of HIV-related criminal laws to ensure the success of population health initiatives and to reduce stigma and discrimination experienced by people living with HIV.