Confusion is a natural part of inquiry-based game-based learning environments (GBLE). While research has explored the relationships between single instances of confusion with motivation and learning, limited work has been done to understand overall confusion dynamics during long-running learning processes and their role in supporting students’ motivation. This study investigated the relationship between students’ confusion with end-of-game motivation and agency using gameplay and facial expression data collected from college students interacting with a GBLE for microbiology. The students were randomly assigned into two conditions; where in the Full Agency condition, students were allowed to move freely in the environment, whereas, in the Partial Agency condition, student actions were limited by a pedagogical scaffolding restricting possible actions to the ones considered most productive for learning. For students in the Full Agency condition (n=58), results showed an inverse relationship between the mean duration of confusion episodes during game-play and self-reported intrinsic motivation following game-play. However, these results were not observed for the Partial Agency condition (n=37), suggesting that pedagogical scaffolding that limits agency may also limit confusion in a GBLE. On the other hand, limiting agency may also limit opportunities to boost intrinsic motivation for students with well-developed self-regulated learning skills. These results suggest promising directions for research into the complex relationships between confusion, motivation, and agency.