Background: Arterial blood puncture is one of the most complex parts of nursing practice. Purpose: Evaluate effect of applying program based learning on nurse's performance and self-efficacy regarding arterial blood puncture. Method: Quasi experimental research design has been utilized in this study. This study was conducted at four hospitals affiliated to ministry of health. A convenient sample of 70 nurses was included. Three tools were utilized for collecting data; first tool was a questionnaire to assess nurses' knowledge regarding arterial blood puncture. Second tool was observational checklist of arterial blood puncture procedure. The last one was arterial blood puncture self-efficacy scale. Results: the study results revealed improvement in nurses' knowledge and practice regarding arterial blood puncture after applying the program based learning. A significant difference was founded between levels of nurses' self-efficacy about arterial puncture after program implementation with p-value of 0.000. While, hospitals where nurses working are associated significantly with total self-efficacy of nurses regarding arterial blood puncture. Conclusion: the current study concluded that the program based learning about arterial puncture improves nurses' knowledge, practice and self-efficacy. Additionally, regular training program regarding arterial blood puncture should be applied in hospitals. Conducting the same study in larger sample is recommended.