Previous research has suggested that abnormalities within the amygdala and prefrontal cortex (PFC) may underlie major depressive disorder (MDD). The contribution of microstructural alterations within these regions in adult MDD is still equivocal. Therefore, seventeen middle-aged medication-free remitted MDD patients and 21 matched never-depressed control subjects underwent structural magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI). Despite comparable amygdala volumes, remitted MDD patients revealed decreased mean diffusivity (MD) and increased fractional anisotropy (FA) within the left amygdala, which may be interpreted as greater cell density and increased number of fibers, respectively. This last notion was supported by probabilistic tractography results, which revealed increased connectivity from the left amygdala to the hippocampus, the cerebellum and the brain stem. Further, altered microstructure as indicated by increased MD possibly reflecting decreased cell density within the medial PFC (mPFC) was found. Taken together, the current DTI study shows that abnormal microstructure and connectivity of the amygdala and mPFC might be key factors in the pathophysiology of MDD that may account for functional changes.