A group of over 200 international scientists came together on April 15 in Sydney, Australia just before the 2012 International Microbicides Conference as a part of a workshop to address the basic concepts and factors that modulate HIV infection at the mucosal surface. The meeting focused on defining the interaction between virus, prevailing host physiology, microbiota, and innate and adaptive immune responses and how they combine to impact the outcome at the moment of potential viral transmission. Speakers examined the biology of HIV entry during transmission, innate and natural antiviral mechanisms at the mucosa, microbicide efficacy, pharmacokinetic, and pharmacodynamics, animal models, and opportunities for combining HIV prevention strategies. Other viral infection models both in vivo and in vitro were considered for the insights they provided into HIV transmission events. The workshop raised important questions that we need to answer to further our basic understanding of host and viral factors influencing HIV transmission to inform the development of novel prevention strategies.