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Background: Unhealthy lifestyle behaviors during young adulthood can have negative impacts on health for the rest of a person’s life. University students are in a transition stage to adulthood and face considerable changes in their life during their time at university. Therefore, the evaluation of their lifestyle and its changes over time using a valid and reliable tool is very important. This study aimed at assessing reliability and validity of the Persian version of the Healthy Lifestyle Scale for University Students (HLSUS). Methods: The HLSUS was developed by Wong et al., based on Pender’s Health Promotion Model, and was translated into Persian using forward/backward translation methods. Content validity of the scale was assessed by a panel of eleven experts. Moreover, Cronbach alpha coefficient was calculated to examine internal consistency. The construct validity was assessed via detecting the factor structure of measurements on a sample of 400 students from different faculties of Tabriz University of Medical Sciences. Scale-item correlations and known group analyses were used to evaluate. convergent and discriminant validity. Results: The HLSUS demonstrated good content validity (content validity index [CVI] values 0.80-1.00 and content validity ratio [CVR] values 0.71-1.00), internal consistency (α = 0.87) and test–retest reliability (intraclass correlation coefficient [ICC] = 0.89). Based on exploratory factor analysis, it was determined that the eight factor solution was optimal for distinguishing the underlying factors. Conclusion: The Persian version of the HLSUS demonstrated initial reliability and validity.