Background: Limited information is available regarding the patient safety culture in Chinese hospitals. This study aims to assess the patient safety culture in Peking University Cancer Hospital and to identify opportunities for improving the organization's safety culture. Methods: A cross-sectional study was conducted in April 2018 and 2019, respectively. Data on patient safety culture were collected from clinical and administrative staffs using the Hospital Survey on Patient Safety Culture (HSOPSC). Results: Twelve composite dimension variables were hierarchically clustered. Three highest positive response dimensions include 'Organizational Learning and continuous improvement' (92.9%), 'Teamwork within units' (89.7%), and 'Hospital management support for patient safety' (83.7%), while 3 lowest positive response dimensions included 'Frequency of events reported' (43.9%), 'Non-punitive response to error' (51.1%), 'Communication openness' (52.2%), and 'Staffing' (53.7%). Compared to the average scores of the United States, the scores of the Peking University Cancer Hospital was significantly lower on 'Communication openness' and 'Frequency of events reported'. After targeted continuous improvement based on results in 2018, all 12 dimensions surprisingly increased in the safety culture conducted in 2019. Conclusion: Inadequate feedback and communications about error and lack of communication openness are key challenges for patient safety in the delivery of care in this hospital. Results of this baseline survey indicate the need for a modified approach and attention to context when designing interventions aimed at improving the safety culture in this organization.