Objective To explore the differences in levels of serum zonulin and claudin-5 between melancholic and atypical depressive disorder and their clinical significance for subtype differentiation. Methods From March 2023 to June 2024， 42 cases each of melancholic and atypical depressive disorder patients meeting the diagnostic criteria of Diagnostic and Statistical Manual of Mental Disorders（ 5th edition） （DSM-5） in the outpatient department of Beijing Huilongguan Hospital were selected as melancholic group and atypical group， and 40 cases of healthy volunteers who had medical checkups in the hospital during the same time period were selected as healthy control group. The severity of the patients' depressive disorder was assessed using the 17-item version of the Hamilton Depression Scale（ HAMD-17）. Serum zonulin and claudin-5 levels were measured in the three groups using enzyme-linked immunosorbent assay to compare the differences between the groups. The differential diagnostic value of the two proteins alone and in combination for the two subtypes of depressive disorder was also assessed using the receiver operating characteristic（ ROC） curve. Results The serum zonulin levels in melancholic， atypical， and healthy control groups were 1 040.69（ 827.23， 1 348.38） ng/ml， 748.38（ 146.46， 954.15） ng/ml， 543.86（ 361.84， 1 015.35） ng/ml， and the claudin-5 levels were 194.20（ 161.79， 268.28） pg/ml， 164.57（ 36.33， 205.77） pg/ml， and 118.60（ 84.86， 190.40） pg/ml， and the differences between the three groups were statistically significant（ all P ＜ 0.05）. The levels of both serum proteins were higher in melancholic group than those in atypical group and healthy control group with statistical differences（ both P＜ 0.05）. There was no statistically significant difference in the levels of the two serum proteins between atypical group and healthy control group（ both P＞0.05）. The area under the ROC curve for serum zonulin to differentiate between melancholic and atypical depressive disorder was 0.741［ 95% confidence interval（ 95%CI）（ 0.635， 0.847）， P ＜ 0.05］， with a sensitivity of 64.3% and a specificity of 76.2%. The area under the ROC curve for serum claudin-5 prediction was 0.679［ 95%C（I 0.564，0.793），P ＜ 0.05］， with a sensitivity of 92.9% and a specificity of 35.7%. The area under the ROC curve for the combined prediction of the two proteins was 0.815 ［95%CI（0.723，0.908），P＜ 0.05）］， with a sensitivity of 85.7% and a specificity of 71.4%. Conclusions Two proteins， serum zonulin and claudin-5， are higher in patients with melancholic depressive disorder than those in patients with atypical depressive disorder and may be effective biomarkers for differential diagnosis of the two subtypes. Zonulin combined with claudin-5 has high clinical value for subtype differential diagnosis.