Background: Video lectures which include the teachers' presence have become increasingly common. As teacher enthusiasm is a nonverbal cue in video lectures, more and more studies are focusing on this topic. However, little research has been carried out on the interactions between teacher enthusiasm and prior knowledge when learning from video lectures. Objectives: We tested whether prior chemistry knowledge moderated the impact of teacher enthusiasm on students' chemistry learning during video lectures. Methods: One hundred and forty-two Grade 7 (low-prior chemistry knowledge) and Grade 9 (high-prior chemistry knowledge) Chinese students engaged with this research. Each group of students was randomised into viewing a video lecture with either a low or high degree of teacher enthusiasm. Outcomes were assessed by attention allocation, learning performance, cognitive load, learning satisfaction and student engagement. Results and Conclusions: Our findings revealed significant benefits of teacher enthusiasm and also showed that prior chemistry knowledge could moderate the impact of teacher enthusiasm on students' attention and cognitive outcomes (cognitive load and transfer). Visual attention mediates the effects on transfer. For students with low prior knowledge, there is more focus on the learning content, lower extraneous cognitive load, and higher transfer scores when watching videos with high levels of teacher enthusiasm; however, students with high prior knowledge do not show these differences. In addition, high prior chemistry knowledge had a significant beneficial impact on the motivational outcomes of the students (satisfaction and engagement). Implications: The results suggest that teacher enthusiasm in a video lecture may affect students' chemistry learning, and students' prior chemistry knowledge should be considered when choosing whether to display teacher enthusiasm.