Objective · To detect the vitamin D2 and D3 levels in the patients with moderate and severe depression. Methods · Eighty-five patients who met the criteria for major depressive disorder were recruited (53 patients with moderate depression, 32 patients with severe depression). Fifty age-matched healthy volunteers were recruited as controls. Serum 25 (OH) D2, 25 (OH) D3 and total 25 (OH) D2/D3 levels were detected by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The risk factors which might influence the severity of depression were screened by Logistic regression analysis. Results · The serum 25 (OH) D2, 25 (OH) D3 and total 25 (OH) D2/D3 levels in the case group was lower than those in the control group (P=0.012, P=0.000, P=0.000). The patients with moderate depression presented significantly lower serum 25 (OH) D3 and total 25 (OH) D2/D3 levels than the controls did (P=0.000), although no significant difference in serum 25 (OH) D2 levels was found between these two groups. As well, the serum 25 (OH) D2, 25 (OH) D3 and total 25 (OH) D2/D3 levels in patients with severe depression were significantly lower than those in patients with moderate depression and controls (P<0.05). The body mass index of severe depression group was much higher than that of moderate depression group and control group (P=0.002). Both overweight/obesity and the concentration of vitamin D may be the major influencing factors of depression severity (P=0.034, P=0.011). Conclusion · Vitamin D2 and D3 deficiency in depressive patients, particularly in those patients with severe depression, was shown in the present study. In addition, overweight/obesity as well as the concentration of vitamin D may exert the significant influence on the severity of depression. Vitamin D supplementation and weight control may be needed to be considered in making therapeutic strategies of major depressive disorder.