The nurses often face moral challenges and conflicts as their duties are performed. In these situations, professional values and religious well-being support their performance within a specific framework and according to defined ethical principles. The purpose of this study is to examine the impact of ethics-based program on Iranian nurses’ professional values and religious well-being. This was an experimental study conducted in Hajar and Ayatollah Kashani hospitals in Shahrekord (Iran) in 2019–2020. The participants included 130 nurses who were selected by convenience sampling and randomly assigned to the intervention and control groups. The outcome variables were professional values and religious well-being of nurses which measured at the beginning of the study, immediately, and two months after the intervention. Data collection occurred at baseline, immediately and 2 months after the intervention by using The Nurses Professional Values Scale-Revised (NPVS-R) and the Spiritual Well-Being Scale (SWBS). The study’s findings revealed a statistically significant difference in mean changes scores related to professional values and religious well-being between the intervention and control groups following the implementation of the intervention. In conclusion, implementation of the ethics-based program can improve the professional values and religious well-being of nurses.