This study explored the effects of prior knowledge and cueing on the learning (retention, transfer, and matching) and mental effort of learners who studied an instructional animation with accompanying narration about photosynthesis. A 4 × 2 between-subjects factorial design with four levels of cueing (no cueing, label cueing, picture cueing, and label and picture cueing) and two levels of prior knowledge (low vs. high) was used. A total of 216 undergraduate students from various majors in a large Southwestern university volunteered to participate in this study. The results revealed no significant effect of cueing on learning or mental effort. However, high prior knowledge learners outperformed low prior knowledge learners on a retention test and reported investing more mental effort than low prior knowledge learners. Although it was not significant, high prior knowledge learners had higher transfer and matching scores when no cues were provided.