AbstractUrbanization is increasing globally, and is associated with stress and increased mental health risks, including for depression. However, it remains unclear, especially at the level of brain function, how urbanicity, social threat stressors, and psychiatric risk may be linked. Here, we aim to define the structural and functional MRI neural correlates of social stress, childhood urbanicity, and their putative mechanistic relevance to depressive illness risk, in terms of behavioral traits and genetics. We studied a sample of healthy adults with divergent urban and rural childhoods. We examined childhood urbanicity effects on brain structure as suggested by MRI, and its functional relevance to depression risk, through interactions between urbanicity and trait anxiety-depression, as well as between urbanicity and polygenic risk for depression, during stress-related medial prefrontal cortex (mPFC) engagement. Subjects with divergent rural and urban childhoods were similar in adult socioeconomic status and were genetically homogeneous. Urban childhood was associated with relatively reduced mPFC gray matter volumes as suggested by MRI. MPFC engagement under social status threat correlated with the higher trait anxiety-depression in subjects with urban childhoods, but not in their rural counterparts, implicating an exaggerated physiological response to the threat context with urbanicity, in association with behavioral risk for depression. Stress-associated mPFC engagement also interacted with polygenic risk for depression, significantly predicting a differential mPFC response in individuals with urban but not rural childhoods. Developmental urbanicity, therefore, appears to interact with genetic and behavioral risk for depression on the mPFC neural response to a threat context.