This study uses longitudinal data from a regional teaching hospital in Taiwan to analyze how nurses with different demographic variables perceive patient safety culture from 2010 to 2016. The results show that nurses with different demographic variables have different perceptions of patient safety from year to year. The supervisor/manager is the most essential variable influencing patient safety culture. Nurses who are supervisors/managers are more satisfied in six dimensions from 2010 to 2016. Experience in an organization is identified as the second essential variable to influence the patient safety culture. Nurses who have much experience in an organization tend to have higher satisfaction in five out of six dimensions of patient safety culture from 2012 to 2016 in general. In contrast, nurses who have less than six months of experience in an organization also have better perceptions. In summary, hospital management needs to pay attention to supervisor/manager and experience in an organization factors, both of which have significant impacts on patient safety culture from nurses’ viewpoints.