Background Reviews of the literature on the health and work environment of ambulance personnel have indicated an increased risk of work-related health problems in this occupation. The aim of this study was to compare health status and exposure to different work environmental factors among ambulance personnel and the core work force in Denmark. In addition, to examine the association between physical and psychosocial work environment factors and different measures of health among ambulance personnel. Methods Data were taken from a nationwide sample of ambulance personnel and fire fighters (n = 1,691) and was compared to reference samples of the Danish work force. The questionnaire contained measures of physical and psychosocial work environment as well as measures of musculoskeletal pain, mental health, self-rated health and sleep quality. Results Ambulance personnel have half the prevalence of poor self-rated health compared to the core work force (5 % vs. 10 %). Levels of mental health were the same across the two samples whereas a substantially higher proportion of the ambulance personnel reported musculoskeletal pain (42% vs. 29%). The ambulance personnel had higher levels of emotional demands and meaningfulness of and commitment to work, and substantially lower levels of quantitative demands and influence at work. Only one out of ten aspects of physical work environment was consistently associated with higher levels of musculoskeletal pain. Emotional demands was the only psychosocial work factor that was associated with both poorer mental health and worse sleep quality. Conclusions Ambulance personnel have similar levels of mental health but substantially higher levels of musculoskeletal pain than the work force in general. They are more exposed to emotional demands and these demands are associated with higher levels of poor mental health and poor sleep quality. To improve work environment, attention should be paid to musculoskeletal problems and the presence of positive organizational support mechanisms that can prevent negative effects from the high levels of emotional demands.