Background: Depression is a common symptom after stroke, but its neural substrates remain unclear. The ascending serotonergic system originates from the raphe nuclei in the brainstem. We hypothesized that depressive disorder due to brainstem infarction is associated with damage to the raphe nuclei. Methods: We prospectively enrolled 19 patients who had the first-ever acute isolated brainstem infarction in an observational cross-sectional study. All patients were evaluated by using the Montgomery.ksberg Depression Rating Scale (MADRS), the clinician-rated version of Apathy Evaluation Scale (AES-C) and Mini-Mental State Examination (MMSE). Depressive disorder was diagnosed according to DSM-5 and MADRS score of 12 or greater. Diffusion tensor imaging and proton density-weighted images were used to identify damage in the raphe nuclei. Accordingly, patients were classified into either the raphe-nuclei damaged or intact group. Prevalence of depressive disorder and the MADRS, AES-C, and MMSE scores were compared between the two groups. Results: Depressive disorder was more frequent in the damaged group (n=6) than in the intact group (n=13) (83% vs. 15%; P=0.01). MADRS scores were higher in the damaged group than in the intact group (mean +/- 1 SD, 17.5 +/- 7.9 vs. 7.0 +/- 4.4; P=0.002), whereas the AES-C and MMSE scores did not differ between groups. Limitations: We did not assess the damage to the ascending projection fibers from the raphe nuclei. Conclusions: Our results suggest that damage to the raphe nuclei underlies depressive disorder due to brainstem infarction, possibly via serotonergic denervation.