Failures in emotion regulation, especially as a result of interpersonal stress, are implicated as transdiagnostic risk factors for psychopathology. This study examines the effects of an experimentally timed targeted interpersonal rejection on emotion reactivity and regulation in typically developing adolescent girls. Girls (n = 33, ages 9–16 years, M = 12.47, SD = 2.20) underwent fMRI involving a widely used emotion regulation task. The emotion task involves looking at negative stimuli and using cognitive reappraisal strategies to decrease reactions to negative stimuli. Participants also engaged in a social evaluation task, which leads participants to believe a preselected peer was watching and evaluating the participant. We subsequently told participants they were rejected by this peer and examined emotion reactivity and regulation before and after this rejection. Adolescent girls evidence greater reactivity via higher self-reported emotional intensity and greater amygdala activation to negative stimuli immediately after (compared with before) the rejection. Self-reported emotional intensity differences before and after rejection were not observed during regulation trials. However, on regulation trials, girls exhibited increased prefrontal activation in areas supporting emotion regulation after compared with before the rejection. This study provides evidence that a targeted rejection increases self-report and neural markers of emotion reactivity and that girls increase prefrontal activation to regulate emotions after a targeted rejection.