Background: During the period of 2016~2018, many efforts were put into promoting the Patient Autonomy Act (PAA) in Taiwan, such as nationally sponsoring advertisements, promoting campaigns, and conducting in-service training programs for healthcare providers. However, no study has examined the impacts of promoting the PAA on healthcare providers. This study was designed to investigate healthcare providers’ knowledge of palliative care and perceptions of palliative care barriers before and after promoting the PAA. Methods: A cross-sectional design was conducted in 2013 and 2018 respectively that used a self-reporting questionnaire to examine healthcare providers’ knowledge of palliative care and perceived barriers to providing palliative care. One hospital recruited 277 healthcare providers in 2013 and 222 healthcare providers in 2018 in another hospital. Statistical analyses involved descriptive statistics for demographic characters. Multivariate linear regression analyses were used to identify predictors of knowledge of and perceived barriers to palliative care. A principal component analysis was to identify the most appropriate factorial structure for the contents of knowledge and perceived barriers to palliative care. Results: Three factors related to knowledge of palliative care were identified in both 2013 and 2018 data: ‘policy, regulation, and promotion’, ‘philosophy and treatments’, and ‘myths and misunderstandings’. Study findings in the two periods were similar. As for barriers to providing palliative care, three factors were identified in 2013 of ‘quality palliative care’, ‘palliative care difficulties’ and ‘messaging and communication’, and in 2018 of ‘messaging and information’, ‘knowledge and attitudes’ and ‘quality palliative care’ were identified. Study findings differed between the two periods. Conclusion: After implementation of the PAA, perceived barriers changed. Policies can better reinforce mitigating strategies—including opportunities for education, shared decision-making, and changes in institutions and care systems. Also, assessing barriers creates important opportunities for further research to address the most critical aspects to improving end-of-life care for patients and their families.