Personality and fitness impact emotional regulation, however the neurobiological mechanisms underlying these relationships are poorly understood. dACC glutamate modulation was quantified during appraisal of emotional images using ¹H fMRS. Additionally, aerobic fitness and trait personality were assessed in participants. The dACC glutamate was lower during positive images compared to neutral images, which was associated with trait openness. Glutamate levels during appraisal of negative emotional images did not differ from neutral images, but was negatively associated with aerobic fitness. These results shed light on the mechanisms that modulate the impact of trait personality and fitness on ACC response during emotional regulation.