The goal of this study was to explore the factors which impact the acceptance of an e-learning course by teachers of elementary and secondary schools in the Czech Republic. The survey involved 228 teachers from 68 different primary and secondary schools. These teachers took part in some of 16 different asynchronous on-line courses aimed at the use of modern technologies in education. It was found that the factors with major impact on the e-learning course acceptance include the voluntary participation of teachers in the course and the positive expectations held before the course start (p < 0.001). Working through the course on one's own and the subjective evaluation of various aspects of the course plays an important role as well (p < 0.001). The factors which had no statistically significant impact (p > 0.05) included the teacher's age, gender, type of school, prior experience with e-learning, individual habits in terms of completing voluntary tasks and taking notes, the involvement in discussions during the course and the time schedule of the course. We conclude that considerable attention should be paid to the psychological aspects of further education of Czech teachers using e-learning.