Background: Patient safety represents a global issue which leads to potentially avoidable morbidity and mortality. This study aimed to determine the inter-professional differences in patient safety culture in a tertiary university hospital. Method: A cross-sectional study using the Safety Attitude Questionnaire (SAQ) self-administered electronically in the English and Malay languages to evaluate safety culture domains. A positive percentage agreement score of 60% was considered satisfactory. Comparisons were made between clinicians, nurses, allied health professionals, ward attendants and support staff. Results: Of 6562 potential respondents, 5724 (80.4%) completed the questionnaire; 3930 (74.5%) women, 2263 (42.9%) nurses, and 1812 (34.2%) had 6-10 years of working experience. The mean overall positive percentage agreement scores were 66.2 (range=31.1 to 84.7%), with job satisfaction (72.3±21.9%) and stress recognition (58.3±25.6%) representing the highest and lowest mean domain scores respectively. Discussion: Differences were observed between all five job categories. Linear regression analyses revealed that the other four job categories scored lower in teamwork, safety culture, job satisfaction, and working conditions compared to nurses. Conclusions: The overall mean SAQ score was above the satisfactory level, with unsatisfactory percentage agreement scores in the stress recognition domain. Interventions to improve patient safety culture should be developed, focusing on stress management.