There are too many new HIV infections globally with 1.8 million persons infected in 2016 alone. Pre-exposure prophylaxis (PrEP) holds potential to decrease new infections and is synergistic with efforts currently in place to achieve an end to the AIDS epidemic in Sub-Saharan African, but uptake is limited. Given its novelty, assessing the beliefs and attitudes of healthcare professionals and members of the community towards HIV transmission and PrEP will be helpful to inform implementation efforts. Study was a random survey of 201 community members and 51 healthcare providers, carried out at multiple community sites in Huye district, Southern Province, Rwanda and at Kigali University Teaching Hospital (KUTH). The study findings are that there are still misconceptions about HIV in the community with some respondents believing that HIV is due to punishment from God (5.4%), poverty (3.0%), smoking cigarettes (1.0%), drinking alcohol (2.0%), punishment from ancestors (1.0%) and witchcraft (1.5%), and that its transmission is by mosquito bites (10.9%), sharing food or drinks with a HIV infected person (6.5%) or as a result of carelessness (47.8%). More than 50% of respondents from both groups had insufficient knowledge regarding PrEP, but expressed some interest in PrEP (82.6% of the respondents from the community and 86.5% of the health workers). However, some healthcare workers felt that promotion of safe sex practices (74.5%), HIV testing and treating HIV infected patients (60.8%) would work better than PrEP to decrease new HIV infections. Barriers to PrEP implementation included perceived stigma, delayed access to prevention services at the health facilities while personal-level concerns included lack of family support, reluctance to take a medication daily and fear of being perceived as having HIV. This study showed that health care workers and community members are willing to utilize PrEP in Rwanda, but many challenges exist including limited knowledge about PrEP, stigma, provider and system level service delivery barriers at health facilities among others. More studies are needed to assess ways of addressing and /or eliminating these barriers.