Introduction: Transformation of patient safety culture towards developing an open culture can be the greatest challenge for achieving a safe healthcare system. This study aimed to carry out a structural analysis of the Persian translation version of a questionnaire assessing patient safety culture. Materials and Methods: The study was conducted to evaluate the Persian translation of patient safety culture questionnaire, developed by the National Patient Safety Agency. The questionnaire includes seven sections and 43 items investigating 12 dimensions of patient safety culture. The reliability of this questionnaire was confirmed with Cronbach's alpha (α>0.8). The questionnaire was distributed among employees of the Fatemeh Zahra Hospital in Najafabad, Iran, 2015. The collected data were analyzed using SPSS 18 and Amos 18. Results: Sufficiency of the sample size, as determined by Kaiser-Meyer-Olkin measure, was 0.809, which was significantly associated with zero; therefore, performing factor analysis was acceptable and justifiable. The value of Bartlett's test was 696, P-value was less than 0.001, and degree of freedom was equal to 91. In the final model, the relative Chi-square was equal to 1.75 and P-value was less than 0.001. Also, parsimony normed fit index, parsimony-adjusted comparative fit index, and root mean square error of approximation were equal to 0.571, 0.621, and 0.065, respectively. Conclusion: Based on the results of fitting indices for the model and the questionnaire used in the present study for assessing patient safety culture, it can be stated that the Persian translation of this instrument is valid and hospitals can use it to monitor patient safety culture improvement.