IMPORTANCE: More than half of all patients with major depressive disorder (MDD) experience a relapse within 2 years after recovery. It is unclear how relapse affects brain morphologic features during the course of MDD. OBJECTIVE: To use structural magnetic resonance imaging to identify morphologic brain changes associated with relapse in MDD. DESIGN, SETTING, AND PARTICIPANTS: In this longitudinal case-control study, patients with acute MDD at baseline and healthy controls were recruited from the University of Munster Department of Psychiatry from March 21, 2010, to November 14, 2014, and were reassessed from November 11, 2012, to October 28, 2016. Depending on patients' course of illness during follow-up, they were subdivided into groups of patients with and without relapse. Whole-brain gray matter volume and cortical thickness of the anterior cingulate cortex, orbitofrontal cortex, middle frontal gyrus, and insula were assessed via 3-Tmagnetic resonance imaging at baseline and 2 years later. MAIN OUTCOMES AND MEASURES: Gray matterwas analyzed via group (no relapse, relapse, and healthy controls) by time (baseline and follow-up) analysis of covariance, controlling for age and total intracranial volume. Confounding factors of medication and depression severity were assessed. RESULTS: This study included 37 patients with MDD and a relapse (19 women and 18 men; mean [SD] age, 37.0 [12.7] years), 23 patients with MDD and without relapse (13 women and 10 men; mean [SD] age, 32.5 [10.5] years), and 54 age- and sex-matched healthy controls (24 women and 30 men; mean [SD] age, 37.5 [8.7] years). A significant group-by-time interaction controlling for age and total intracranial volume revealed that patients with relapse showed a significant decline of insular volume (difference, -0.032; 95%CI, -0.063 to -0.002; P = .04) and dorsolateral prefrontal volume (difference, -0.079; 95%CI, -0.113 to -0.045; P < .001) from baseline to follow-up. In patients without relapse, gray matter volume in these regions did not change significantly (insula: Difference, 0.027; 95%CI, -0.012 to 0.066; P = .17; and dorsolateral prefrontal volume: Difference, 0.023; 95%CI, -0.020 to 0.066; P = .30). Volume changes were not correlated with psychiatric medication or with severity of depression at follow-up. Additional analysis of cortical thickness showed an increase in the anterior cingulate cortex (difference, 0.073 mm; 95%CI, 0.023-0.123 mm; P = .005) and orbitofrontal cortex (difference, 0.089 mm; 95%CI, 0.032-0.147mm; P = .003) from baseline to follow-up in patients without relapse. CONCLUSION AND RELEVANCE: A distinct association of relapse in MDD with brain morphologic features was revealed using a longitudinal design. Relapse is associated with brain structures that are crucial for regulation of emotions and thus needs to be prevented. This study might be a step to guide future prognosis and maintenance treatment in patients with recurrent MDD.