Public health nurses (PHNs) use many interventions to prevent illness and promote the health of populations. Unfortunately, generating evidence regarding PHN practice is not explicitly identified as a research priority area of the major national funding agencies. Nor has PHN, as a profession, had a strong enough research agenda to drive practice improvement on a population-level and to drive funding to support such areas of research. To further advance the science needed to guide PHN practice, a national conference to set the research agenda was held in October 2010 with grant support from the Agency for Healthcare Research and Quality. The conference was part of a multimethod, participatory, multistage approach taken to generate the final research priority themes and corresponding priority research questions. The process yielded four high priority PHN research themes: PHN intervention models, Quality of population-focused PHN practice, Metrics of/for PHN, and comparative effectiveness and PHN outcomes. As the agenda is adopted by funding agencies, researchers, and practice-based partners, a more focused program of research will produce evidence that can guide population-focused PHN practice.