The anterior cingulate cortex (ACC) and insula are important neural substrates for the integration of cognitive, emotional, and physiological information, as well as the coordination of responses to anticipated stimuli. Increased neural activation within these structures has been observed in individuals with anxiety and depressive disorders. Selective serotonin reuptake inhibitors (SSRIs) are among the most effective and frequently prescribed anxiolytic agents, yet it is not known whether ACC or insula underlie the effects of these drugs. We examined whether subchronic administration of a SSRI to healthy volunteers attenuates activation in ACC or insula during anticipation, an important emotional process underlying anxiety. Support for this hypothesis would help to understand where and by what process SSRIs may exert beneficial effects as anxiolytics and would provide further mechanistic evidence for functional magnetic resonance imaging (fMRI) as a biomarker for the development of anxiolytics. Fifteen volunteers participated in a double-blind, placebo-controlled, randomized cross-over study. Participants completed a pleasant and aversive picture-cued anticipation task during fMRI after taking either escitalopram (10 mg) or placebo for 21 d. We found that escitalopram significantly decreased activation in bilateral posterior and middle insula during the anticipation condition irrespective of stimulus valence and in medial prefrontal and ACC during anticipation of aversive vs. pleasant images. Reduced insular and ACC activation in healthy controls during anticipation may be integral to the therapeutic efficacy of SSRIs and may provide a mechanistic approach for the use of pharmaco-fMRI in the identification of novel pharmacotherapeutic agents in patient populations.