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Background: The purpose of this study was to investigate the effect of Appendectomy Surgical Simulation software (game-based) on the self-efficacy of undergraduate students in the operating room. Methods: In this single-blind quasi-experimental study, 70 second semester undergraduate students in Isfahan City, Iran, were selected using convenience sampling method. After obtaining satisfaction, they were assigned to intervention and control groups randomly. Simulation software (game-based) and the holding practice and lecture class (routine method) were used in intervention and control groups, respectively. Then, the data were collected using a researcher-administered questionnaire of self-efficacy which validity and reliability were confirmed. Data were then analyzed using SPSS software and descriptive and analytical tests with significance level of less than 0.05. Findings: The average score of self-efficacy in the intervention group (62.97 ± 1.48) was higher than the control group (48.63 ± 2.67), and the difference was statistically significant (P < 0.05). Moreover, there were no significant relationship between demographic characteristics and self-efficacy (P > 0.05). Conclusion: Appendectomy Surgery Simulation software can promote students' self-efficacy. Therefore, it is recommended to be used as a supplementary training method for teaching the operating room students.