Background: Sepsis has a high mortality. Early recognition and timely treatment are essential for patient survival. The aim of this study is to examine the factors that influence the knowledge and recognition of systemic inflammatory response syndrome (SIRS) criteria and sepsis by emergency department (ED) nurses. Methods: A prospective, multi-center study including 216 ED nurses from 11 hospitals and academic medical centers in The Netherlands was conducted in 2013. A validated questionnaire was used to evaluate ED nurses' knowledge about SIRS and sepsis. Questions about demographic characteristics were also included, to investigate factors that may contribute to the knowledge about SIRS and sepsis. Results: The mean total score was 15.9 points, with a maximum possible score of 29 points. ED nurses employed at hospitals with a level 3 intensive care unit (ICU) scored significantly higher than their colleagues employed at hospitals with a level 1 or 2 ICU. Recently completed education in sepsis was associated with a higher score. The employees in low ICU level hospitals who reported recent education did not score significantly lower than their ICU level 3 colleagues. ED nurses over the age of 50 scored significantly lower than their younger colleagues. Conclusions: The knowledge of ED nurses concerning SIRS and sepsis rises proportionally with the level of ICU in hospitals. Recent education in sepsis raises knowledge level as well. We recommend that when there is a low exposure rate to SIRS and sepsis, more emphasis should be placed on regular education.