We examined the impact of autonomy on college students’ emotions and learning with a game-based learning environment (GBLE). 96 undergraduate students participated in the study. Participants were randomly assigned to one of three experimental conditions based on the level of control granted during gameplay. Results revealed participants in the partial agency condition achieved the highest proportional learning gains (PLG), after controlling for session duration. There was a positive correlation between evidence scores of four emotions and PLG within the partial agency condition. A stepwise multiple regression showed anger as the best and sole predictor of PLG. Implications include understanding the role of autonomy and emotions during learning and problem-solving with GBLEs designed to foster scientific thinking in STEM. The current study suggests that although GBLEs offer significant learning benefits they also induce several basic and learning–centered emotions that can either facilitate or inhibit learning gains which require further examination.