Background: Although adding embodied instructors on the screen is considered an effective way to improve online multimedia learning, its effectiveness is still controversial. The level of realism of embodied on-screen instructors may be an influencing factor, but it is unclear how it affects multimedia learning. Aims: We explored whether and how embodied on-screen instructors rendered with different levels of realism in multimedia lessons affect learning process and learning outcomes. Samples: We recruited 125 college students as participants. Methods: Students learned about neural transmission in an online multimedia lesson that included a real human, cartoon human, cartoon animal or no instructor. Results: Students learning with cartoon human or cartoon animal instructors tended to fixate more on the relevant portions of the screen and performed better on retention and transfer tests than no instructor group. The real human group fixated more on the instructor, fixated less on the relevant portion of the screen and performed worse on a retention test in comparison to the cartoon human group. Fixation time on the instructor fully mediated the relationship between instructor realism and retention score. Conclusions: The addition of embodied on-screen instructors can promote multimedia learning, but the promotion effect would be better if the embodied instructor was a cartoon animal or cartoon human rather than a real human. This suggests an important boundary condition in which less realism of on-screen embodied instructors produces better learning processes and outcomes.