This study aimed to investigate the impact of scaffolding prompt questions on learners' self-regulated learning about the Nature of Science (NOS) in a hypermedia environment. In this study, mixed methods research design was employed. Sixty-four pre-service science teachers (n=64) were randomly assigned to the experimental group (N:33, scaffolding for self-regulation) and the control group (N:31, no scaffolding for self-regulation). Both groups were trained on how to use hypermedia while learning about NOS. Participants in the experimental group were scaffolded regarding how to regulate learning with hypermedia, whereas participants in the control were not given any instruction about self-regulation. MSLQ and think-aloud protocols were used to measure participants' self-regulation behaviors. Also, we collected data via VOSTS questionnaire to identify any changes in participants' understandings about NOS from pre-test to post-test. The findings that emerged from MSLQ showed that the experimental group performed better self-regulation behaviors than the control group. Also, it was found that participants in the experimental group used several effective self-regulated behaviors which seemed to foster their learning. By contrast, participants in the control group were not effective at regulating their learning. According to the control group, the analyses of post-tests of VOSTS indicate that the experimental group achieved a more informed understanding of NOS. Implications for enhancing our understandings of how learners self-regulate their learning process and what assistance they need are presented.