Background We aimed to explore (1) the influence of healthcare professionals' (HCPs') specialty, profession, gender and length of employment on their perception of six dimensions of patient safety culture (PSC) and (2) the relation between these characteristics and the two dimensions of safety climate and perception of management. Methods In a cross-sectional study, a Danish version of the Safety Attitudes Questionnaire was sent to all HCPs at a large regional hospital organisation. This included hospitals, the Emergency Services, the Regional Pharmacy and the Centre for Diabetes corporations. A total of 30 230 HCPs received the survey. Differences between specialties, professions, gender and years of employment were tested for each dimension of PSC. Differences in mean attitude scores were tested using analysis of variance and differences in having a positive attitude were tested using logistic regression. Results In total, 15 119 (50%) HCPs returned the survey. Significant differences are seen across hospitals and corporations for all dimensions of PSC. The proportion of HCPs with a positive attitude was largest regarding job satisfaction (74.8%) and lowest regarding perception of management (43.9%). Significant differences are seen in physicians' and nurses' perception of PSC in the different specialties within all dimensions of PSC except for the dimension of recognition of stress. Significant differences in positive perception of teamwork climate are seen between anaesthesiologists' (69.4%) and surgeons' (41.7%). No significant gender differences were found between physicians' and nurses' perception of safety climate and of management. In addition, we found an influence of years of employment on PSC. Discussion Significant differences were found in HCPs' perception of PSC between corporations, specialties and professions. The lowest proportion of HCPs with a positive perception of PSC was found within the dimensions of safety climate and perception of management. These differences may have implications for teamwork and patient safety.