This update of the clinical protocol and therapeutic guidelines for pre-exposure prophylaxis (PrEP) in Brazil broadens the eligibility criteria to all sexually active adults and adolescents at increased risk of HIV acquisition, recommends an initial loading dose of two tablets of tenofovir disoproxil fumarate/emtricitabine (TDF/FTC) on the first day of oral PrEP use, and includes changes in laboratory follow-up.