Drawing on cognitive theories, this study intends to investigate the effects of explicit visual cues which have been proposed as a critical factor in facilitating understanding of biological images. Three diagrams from Taiwanese textbooks with implicit visual cues, involving the concepts of biological classification systems, fish taxonomy, and energy pyramid, were selected as the reading materials for the control group and reformatted in tree structure or with additional arrows as the diagrams for the treatment group. A quasi-experiment with an online reading test was conducted to examine the effect of the different image conditions on reading comprehension of the two groups. In total, 192 Taiwanese participants from year 7 were assigned randomly into either control group or treatment group according to the pre-test of relevant prior knowledge. The results indicated that not all explicit visual cues were significantly efficient. Only the explicit tree-structured diagrams cued significantly the key concepts of qualitative class-inclusion, parallel relations, and fish taxonomy. Meanwhile the effect of indexical arrows was not significant. The inconsistent effect of tree structure and arrows might be related to the extent of image reformation in which the tree-structured diagrams had undergone radical change of knowledge representation; meanwhile, the arrows had not changed the diagram structure of energy pyramid. The factor of prior knowledge was essential in considering the influence of image design as the effect of diagrams was very different for low and high prior knowledge students. Implications are drawn for the importance of visual design in textbooks.