The purpose of this study was to investigate the effects of cueing and prior knowledge on learning and mental effort of students studying an animation with narration. This study employed a 2 (no cueing vs. visual cueing) × 2 (low vs. high prior knowledge) between-subjects factorial design. The results revealed a significant interaction effect between prior knowledge and cueing on learning. Low prior knowledge learners had higher scores after studying an instructional animation with visual cues, compared to those who studied the same instructional animation without visual cues. Conversely, when cues were not provided, high prior knowledge learners outperformed those high prior knowledge learners who studied with the cued version of an instructional animation. These results indicated that the effects of cueing in an instructional animation change depending on the learners' level of prior knowledge. Specifically, low prior knowledge learners benefited more when visual cues were provided, whereas cues did not facilitate learning for high prior knowledge learners.