Neurofunctional mechanisms underlying cognitive behavior therapy (CBT) are still not clearly understood. This functional magnetic resonance imaging (fMRI) study focused on changes in brain activation as a result of one-session CBT in patients suffering from spider phobia. Twenty-six female spider phobics and 25 non-phobic subjects were presented with spider pictures, generally disgust-inducing, generally fear-inducing and affectively neutral scenes in an initial fMRI session. Afterwards, the patients were randomly assigned to either a therapy group (TG) or a waiting list group (WG). The scans were repeated one week after the treatment or after a one-week waiting period. Relative to the non-phobic participants, the patients displayed increased activation in the amygdala and the fusiform gyrus as well as decreased activation in the medial orbitofrontal cortex (OFC) during the first exposure. The therapy effect consisted of increased medial OFC activity in the TG relative to the WG. Further, therapy-related reductions in experienced somatic anxiety symptoms were positively correlated with activation decreases in the amygdala and the insula. We conclude that successful treatment of spider phobia is primarily accompanied by functional changes of the medial OFC. This brain region is crucial for the self-regulation of emotions and the relearning of stimulus-reinforcement associations.