Prenatal cocaine exposure (PCE) is associated with long-term and negative effect on arousal regulation. Recent neuroimaging studies have examined brain mechanisms related to arousal dysregulation with cross-sectional experimental designs; but longitudinal changes in the brain, reflecting group differences in neurodevelopment, have never been directly examined. To directly assess the interaction of PCE and neurodevelopment, the present study used a longitudinal design to analyze functional magnetic resonance imaging (fMRI) data collected from 33 adolescents (21 with PCE and 12 non-exposed controls) while they performed the same working memory task with emotional distracters at two points in time. The mean age of participants was 14.3 years at time_1 and 16.7 years at time_2. With confounding factors statistically controlled, the fMRI data revealed significant exposure-by-time interaction in the activations of the amygdala and default mode network (DMN). For the control adolescents, brain activations associated with emotional arousal (amygdala) and cognitive effort (DMN) were both reduced at time_2 as compared to that at time_1. However, these activation reductions were not observed in the PCE group, indicating persistently high levels of emotional arousal and cognitive effort. In addition, correlations between longitudinal changes in the brain and in behavior have shown that adolescents with persistently high emotional arousal were more likely in need of high cognitive effort; and their cognitive performance was more likely to be affected by distractive challenges. The present results complement and extend previous findings from cross-sectional studies with further evidence supporting the view of PCE associated long-term teratogenic effects on arousal regulation.