Background: In order to prove their commitment to the nursing profession, nurses need to base their professional activities on certain acknowledged values. Although a large number of studies have addressed professional values in nursing, only a few studies are available on the identification and comparison of nurses’, nursing students’, and nursing instructors’ understanding of such values. Objective: The study aims to compare nurses’, nursing students’, and nursing instructors’ perception of nursing professional values. Research design: In this descriptive-comparative study, data were (...) collected using Weis and Schank’s Nurses’ Professional Values Scale–Revised. The data were analyzed using the SPSS statistical software (v 22). Participants and research context: A total of 299 nurses, 341 nursing students, and 100 nursing instructors from multisite, 20 different wards from 3 university hospitals and associated nursing schools located in the cities of Shiraz, Fasa, and Jahrom in Fars province, participated in 2016. Ethical considerations: The Institutional Review Board of the researchers’ primary university has verified that the study complies with research ethics. Findings: The total mean scores of the nurses’, nursing students’, and nursing instructors’ perception were found to be 4.23 (0.44), 3.92 (0.50), and 4.34 (0.35), respectively, in the domain of justice—this domain was the subjects’ top priority—and 3.40 (0.56), 3.29 (0.49), and 3.55 (0.36), respectively, in the domain of activism—this domain was attached the least importance by the subjects. There were significant differences across the three groups’ perception in all of the dimensions of professional values ( p < 0.001). Discussion: The three study groups’ overall mean scores fall within the range of relatively important or important. Several studies show the same results, but there are still controversies in this regard. Conclusion: There is need for plans to increase nurses’ awareness of certain professional duties and improve their professional performance in all areas alongside their care duties. (shrink)