The present study examined whether students' prior knowledge moderated the effects of their motivation compositions on learning performance (ie, retention and transfer) and interaction (ie, interpersonal brain synchronization (IBS) and behavioural pattern) in learning from video lectures. The results confirmed the benefits of the composition of two high motivation students on their knowledge transfer. The results also showed that students' prior knowledge had a moderating effect on interaction. For low prior knowledge students, high motivation composition increased their IBS in the temporoparietal junction-inferior parietal lobule (TPJ-IPL), temporoparietal junction-supplementary motor area (TPJ-SMA), inferior parietal lobule-supplementary motor area (IPL-SMA) and anterior prefrontal-anterior prefrontal cortex (aPFC-aPFC) regions during co-explaining and enhanced their regulating-related behavioural sequences and decreased disagree-related behavioural sequences. However, for high prior knowledge students, a high motivation dyad composition decreased their IBS in the supramarginal gyrus-anterior prefrontal cortex (SMG-aPFC) while co-viewing the video lecture, and in the TPJ-IPL, TPJ-SMA, IPL-SMA and SMG-aPFC during co-explaining, cognitive communication, argumentation-related and regulating-related behavioural sequences but enhanced off-task-related behavioural sequences. Our findings suggest that instructors should encourage high motivation students to co-view video lectures, and scaffold students with low prior knowledge to enhance regulating-related behavioural sequences and scaffold students with high prior knowledge and high motivation to enhance cognitive communication, argumentation-related and regulating-related behavioural sequences and decrease off-task-related behavioural sequences.