The goal of this study was to examine the association between affective development, puberty, and gender using the startle reflex as a marker of defensive mechanisms. Thirty-one male and thirty-five female adolescents aged ten to thirteen participated in a prospective study with up to five assessments. Longitudinal analyses revealed a significant effect of sex, with girls showing stronger fear-potentiation at all pubertal stages. Post hoc tests revealed that fear-potentiation increased in girls but not boys over the course of puberty. Furthermore, baseline startle decreased over the course of puberty. Because age was included as a covariate in all analyses, the puberty effect cannot be accounted for by age. To the best of our knowledge, this study provides the first evidence for a significant increase in fear-potentiated startle across the pubertal transition. Attribution of these changes to pubertal status rather than age has important implications for our understanding of the neurobiology of anxiety and affect regulation.