Conceptual understanding is an essential competency that students need to develop in biology learning, particularly when studying complex and abstract topics such as the human locomotor system. This study aimed to analyze the effect of implementing the Discovery Learning model assisted by the BioDigital Human 3D Web on students’ conceptual understanding of the human locomotor system and to examine students’ responses to the use of the learning media at SMA Negeri 2 Maumere. This study employed a quantitative approach using a quasi-experimental method with a Nonequivalent Control Group Design. The population consisted of all Grade XI science students, while the samples were selected using purposive sampling and comprised an experimental class and a control class. Data were collected using a conceptual understanding test administered as a pretest and posttest, along with a student response questionnaire. The data were analyzed using descriptive statistics and an Independent Samples t-test. The results showed a significant difference between the experimental and control classes, as indicated by a significance value of 0.000 (p < 0.05). The experimental class achieved a higher mean posttest score (83.57) than the control class (54.27). Furthermore, students’ responses to the use of the BioDigital Human 3D Web reached 85.46%, indicating a very positive response. Therefore, integrating the Discovery Learning model with the BioDigital Human 3D Web effectively improves students’ conceptual understanding of the human locomotor system and provides a more interactive and meaningful learning experience.