Purpose: Gender mainstreaming is an important concept and strategy for promoting gender equality. It promotes gender sensitivity and improves the quality of health care. The purpose of this study was to apply the concept of gender mainstreaming to a nursing training course to explore its effectiveness in improving the gender stereotypes and the gender attitudes of nursing students. Methods: We used a quasi-experimental research design with purposive sampling to recruit students in a continuing education nursing program. We provided a training course to 34 students in the experimental group for 9 weeks (4 hours per week). The 47 students in the control group continued their regular course. We administered gender stereotype and gender role attitude scales, and collected students' reflections on learning from experimental group. We conducted paired t-tests, and repeated measures two-way ANOVAs. We used content analysis to review their learning reflections. Results: At the end of the training course, the experimental group held significantly fewer gender stereotypes than the control group, and their gender role attitudes became significantly more positive than the control group's. Their social rights and obligations scores, competence score, and performance also significantly increased as compared to before the course. Based on the content analysis of students' written reflections, four themes were extracted from text that supported the results of the quantitative data. Conclusion: A gender mainstreaming course can change the gender stereotypes and gender roles of nursing students in Taiwan. We suggest that teachers develop curriculum to help students' learn in-depth thinking about and reflection on gender awareness.