A bioinformatics laboratory experiment was designed to help undergraduate students learn RNA structure and function in biochemistry courses. In this laboratory experiment, students used 5S rRNAs as material to predict secondary structure and visualize three-dimensional (3D) structure. Students first downloaded molecular information from the 5S rRNA Database and the Protein Data Bank, then predicted secondary structures using the RNA prediction tools Mfold and LocARNA, and last observed 3D structures with the visualization tool Jmol. Students gained basic bioinformatics skills and enhanced their understanding about RNA by using these sequential operations and RNA bioinformatics tools. Their learning outcomes were assessed by pre- and postlaboratory examinations as well as the corresponding questionnaire. The evaluation results showed that this bioinformatics laboratory experiment is suitable and helpful for learning RNA structure and function.