Various authors have investigated students’ interpretations of biochemistry visualizations, but none to our knowledge have compared the intentions of the visualizations’ design with students’ interpretations. This study contrasts an animator’s educational intentions for an animation visualizing ATP (adenosine triphosphate) synthesis, catalysed by the enzyme Fo/F1-ATP-synthase, with 43 university students’ interpretation of the animation. The aim was to identify symbolic expressions in the animation and assess how well they succeed or fail to communicate the intended learning object. We explored the animators’ intentions in a semi-structured interview. To analyse how the students observed and interpreted the animation we first collected individual written responses in a combined worksheet and questionnaire from the students who were using the animation as a thinking tool. Immediately thereafter we also recorded the students’ argumentation and reasoning in group discussions based on the same questions.’ In total, six key facets intentionally illustrated by the animator were successfully interpreted by the students: 1) The dynamics and movement in the protein 2) The conformational changes induced, 3) The driving force of the process (the proton gradient), 4) The causal sequence (coupling) in the process, 5) The cellular context and nature (protein) of the main actor and 6) The energy transfer. Four of the symbolic expressions chosen by the animator helped the students to interpret these facets of the process. Students’ successfully discerned the conformational change in the protein, the rotation of the catalytic part of the protein and the connection between the proton gradient and ATPsynthesis due to the transitory movement depicted in the animation. In addition, use of a ribbonmodel helped students to intuitively grasp that a protein was involved and the sub-microscopic nature of the process. However, a flash intentionally used to indicate the energy transfer associated with the formation of the phosphodiester bond, was misinterpreted by the students as a release of energy, instead of an energy transformation from mechanical to temporarily stored energy in a chemical bond. Further, only five students were able to predict the reversibility of the process from the animation.