Aim To compare Spanish nursing students' and registered nurses' perception of professional values from the perspective of gender and professional experience. Background Nursing has traditionally been a female profession; however, the analysis of professional nursing values from a gender perspective has received little attention. The integration of professional values typically begins during academic life and is, thereafter, reinforced during professional activities. Methods A descriptive cross‐sectional study was conducted with 159 students (97.4% females) and 386 registered nurses (86.8% females) from primary healthcare centres, hospitals and management positions within the public health system. The validated Spanish adaptation of the Nurses Professional Values Scale was used. This instrument measures three dimensions: ethics, commitment and professional mastery. Descriptive statistics were used to analyse the data, together with bivariate analysis using the chi‐squared test. Results Females granted more importance to professional values. Important gender differences among students were detected. Ethical values were considered to be the most important for both students and nurses, followed by professional mastery and commitment. The importance given to values progressively decreased in the groups under study: students, less‐experienced nurses and expert nurses (in decreasing order). Conclusions Significant gender differences exist with regard to professional nursing values. The values related to professional commitment and mastery should be integrated and promoted during academic education and specifically focus on male students' perceptions. As professional experience increases, the importance attributed to professional values decreases. Implications for Nursing Policy Professional nursing values should be promoted among universities, health services and professional organizations, encouraging their dissemination, implementation and evaluation.