Many researchers have called for implementation of active learning practices in undergraduate science classrooms as one method to increase retention and persistence in STEM, yet there has been little research on the potential increases in student anxiety that may accompany these practices. This is of concern because excessive anxiety can decrease student performance. Levels and sources of student anxiety in three introductory biology lecture classes were investigated via an online survey and student interviews. The survey (n = 327) data revealed that 16% of students had moderately high classroom anxiety, which differed among the three classes. All five active learning classroom practices that were investigated caused student anxiety, with students voluntarily answering a question or being called on to answer a question causing higher anxiety than working in groups, completing worksheets, or answering clicker questions. Interviews revealed that student anxiety seemed to align with communication apprehension, social anxiety, and test anxiety. Additionally, students with higher general anxiety were more likely to self-report lower course grade and the intention to leave the major. These data suggest that a subset of students in introductory biology experience anxiety in response to active learning, and its potential impacts should be investigated.