Public health regulations practices surrounding sex work and their enforcement can have unintended consequences for HIV and sexually transmitted infection (STI) prevention and care among sex workers. This analysis was based on qualitative in-depth (n = 33) and focus groups interviews (n = 20) conducted with migrant female sex workers in Tecún Umán and Quetzaltenango, Guatemala, and explored the implementation of sex work regulations and related consequences for HIV prevention and care among migrant sex workers. Sex work regulations were found to have health-related benefits (e.g., access to HIV/STI testing) as well as negative impacts, such as abuse by police and harassment, detention/deportation of migrant sex workers. Whereas public health regulations may improve access to HIV/STI testing, their implementation may inadvertently jeopardize sex workers' health through unintended negative consequences. Non-coercive, evidence-based public health and sex work policies and programs are needed to expand access to HIV/STI prevention and care among migrant sex workers, while protecting their dignity and human rights.