Background and Aim: Improper administration of oxygen as an expensive drug has dangerous side effects. Nurses are responsible for performing this therapy and even prescribing it in emergencies. This study aimed to determine the effect of two methods of mastery learning and lectures on the oxygen therapy knowledge and practice of nurses in a teaching hospital in southern Iran.Materials and Methods: This quasi-experimental study was conducted on 50 nurses working in a teaching hospital in southern Iran. The participants were selected using convenience sampling and randomly divided into intervention and control groups. The members of the intervention group attended 4 oxygen therapy master learning sessions, while the members of the control group were trained using the lecture method for 4 sessions. The instruments used for data collection were a demographic information questionnaire and the researcher-made nurses’ oxygen therapy knowledge and practice inventories that were completed by the participants in both groups. The data were collected before the intervention and one week and three months after the intervention and analyzed using SPSS software with descriptive and inferential statistics.Results: The findings indicated that the nurses had poor oxygen therapy knowledge and practice before the intervention. Furthermore, there was no significant difference between the two groups in terms of the mean scores of oxygen therapy knowledge and practice before the intervention. It was also shown that oxygen therapy knowledge and practice were significantly improved one week and three months after the learning mastery intervention in the participants in the intervention group (P < 0.001). In contrast, only the oxygen therapy knowledge of the members of the control group showed a significant increase three months after the intervention compared to the pre-intervention stage (P = 0.04). Conclusion: The results of the present study showed that the mastery learning model is an effective model for improving and maintaining the nurses’ oxygen therapy knowledge and practice. Since nurses’ optimal practice improves the quality of care and increases patient safety, mastery learning can be used as an effective training technique to improve nurses’ oxygen therapy practice.