In regards to the urgency of knowledge management, commercializing it and changes in the nature of economical production today, universities as the major institution of producing knowledge, play a new role in economic development. 
To materialize this role, they need to be transformed into entrepreneurial universities. In such universities, managers are the main players in decision making; therefore, they should possess special abilities and capabilities including entrepreneurial skills. Consequently, it is essential to motivate these managers to bring their capabilities into action. Since the development of entrepreneurial behavior is affected by economic entrepreneurial climate, on the one hand, and organizational entrepreneurial climate, on the other, two sets of internal/external factors were determined in this research and their effects on 
entrepreneurial behavior of middle managers of selected universities of Iran were studied. The present study is a correlational survey and has used cross-sectional method. The statistical population consisted of middle managers of Sharif University, Beheshti University, Amir Kabir University, Science and Technology University and Kharazmi University. The sample size was 209, estimated by Cochran’s formula. In order to analyze the data, factor analysis method, structural equations, and Friedman’s non-parametric test were used. Results show that such internal factors as freedom of action, working independence, time availability and organizational boundaries have positive and significant effect on entrepreneurial behavior of middle managers of selected universities, but factors like bonuses, reinforcement, and monetary, fiscal and currency policies of government have negative and significant effect on entrepreneurial behavior. From among all variables studied, top management support and business policies show no significant effect on entrepreneurial behavior.