The present study is conducted to implement the newly developed questionnaire to assess the readiness of academics for Implementation of e-learning programs. The questionnaire was developed and its validity and reliability were evaluated. Then a cross-sectional study was conducted including 907 academics in three categories (faculties, students, and employees) of Shiraz University of Medical Sciences. Data were analyzed in SPSS-16 using descriptive and inferential statistics. The results of the implementation of the questionnaire showed a significant relationship between e-learning knowledge and participants' position in the university, as the professors and students had higher levels of this knowledge compared to the university employees (P<0.001). There was a significant relationship between e-learning knowledge and an academic degree in the study subjects (P=0.004). Skills and attitude were also significantly related to the variables of position, academic degree, and place of residence and hours of internet use. Skills, knowledge and attitude were all related to computer and internet access and language used on the internet.