Academic self-efficacy encompasses judgments regarding one's ability to perform academic tasks and is correlated with achievement and persistence. This study describes changes in biology self-efficacy during a first-year course. Students (n = 614) were given the Biology Self-Efficacy Scale at the beginning and end of the semester. The instrument consisted of 21 questions ranking confidence in performing biology-related tasks on a scale from 1 (not at all confident) to 5 (totally confident). The results demonstrated that students increased in self-efficacy during the semester. High school biology and chemistry contributed to self-efficacy at the beginning of the semester; however, this relationship was lost by the end of the semester, when experience within the course became a significant contributing factor. A proportion of high- and low-achieving (24 and 40%, respectively) students had inaccurate self-efficacy judgments of their ability to perform well in the course. In addition, female students were significantly less confident than males overall, and high-achieving female students were more likely than males to underestimate their academic ability. These results suggest that the Biology Self-Efficacy Scale may be a valuable resource for tracking changes in self-efficacy in first-year students and for identifying students with poorly calibrated self-efficacy perceptions.