The purpose of this study was to explore the impact of authentically based instruction using the 5E model as the structure for lesson delivery on student emotions and the extent to which the students perceived that the teacher was facilitating mastery goals. Participants were 241 sophomore and junior biology students in three urban high schools in a large city in the Midsouth region of the United States. Two conditions occurred: a lesson designed using authentic 5E model, while the other was a text-based lesson on the topic of photosynthesis. The results suggest that the authentic 5E instruction increased interest and enjoyment and decreased anxiety, hopelessness, frustration, confusion, and boredom as compared to the text-based instruction. Furthermore, the students in the authentic group perceived that the teacher facilitated more mastery goals than the comparison group. Finally, we found that the change of student perception in teacher mastery had a predictive relationship with change in positive and negative emotions. Implications of this study are that authentic instruction can lead to positive academic outcomes and that perceived teacher mastery can have a positive influence on student emotions, which in turn can generate motivation and achievement in high school science courses.