Introduction: Today, e-learning is considered one of the efficient learning contexts in continuing medical education (CME). Participation in e-learning is dependent on learner’s level of preparedness. Therefore, this study aimed to determine CME learners’ knowledge, attitude, ability and preparedness in relation to e-learning. Methods: In this descriptive cross-sectional study, 300 learners attending 2014 autumn-winter CME programs in Isfahan University of Medical Sciences were selected by means of convenience sampling. Data were collected through a researcher-made questionnaire which was completed in continuing education sessions. The data were analyzed using descriptive and inferential statistics (independent t-test, Pearson and Spearman correlation coefficients). Results: The mean scores were obtained for ability and knowledge (37.23±8), attitude (74.18±3.4) and preparedness (60.22±7.8). Findings showed that there was a significant relationship between computer and internet usage time and learner’s ability and knowledge, attitude and preparedness for e-learning (P<0.001). There was also a significant relationship between learners’ knowledge and ability and their attitude and preparedness (P<0.001). Gender was found to have no effect on ability and knowledge (p=0.08), attitude (p=0.134) and preparedness (p=0.54) for e-learning. Conclusion: The results of this study showed that despite having the features of attitude and preparedness, CME learners did not have the necessary knowledge for receiving information electronically. It seems that authorities should take necessary measures to implement this teaching method in order to promote learners’ knowledge.