Long-term negative affect in adolescence is associated with impairment in quality of life, interpersonal function, and social adaptation. Although physical exercise could decrease negative emotion, the underlying mechanism remains largely unknown. Acute exercise with controlled intensity might be a good experimental paradigm to unravel the potential neural mechanisms underlying the effects of physical exercise on negative affect. In this study, twenty-three males in late adolescence were randomly assigned to acute exercise group (AG) or control group. The experiment contained pre-test and post-test session interleaved with 30-min moderate-intensity exercise or seated rest. In each session, a resting-state fMRI scanning was conducted followed by completing Positive and Negative Affect Schedule and Profile of Mood State. Bilateral amygdala was used as seed region to calculate t voxel-wised functional connectivity (FC) of amygdala to whole brain. The results demonstrated, for the first time, that AG exhibited increased FC between right amygdala and right orbital frontal cortex. Significantly decreased negative affect was also observed in AG. Moreover, the increased rOFC-amygdala FC was also associated with the decreased depression score. Our findings suggest that exercise-induced decreased negative affect might be modulated by functional interactions of amygdala with both cognitive control and limbic networks, which offers a meaningful insight for clinical treatment and prevention of emotional disorders in late adolescence.