AbstractDisturbed gut microbiota is a potential factor in the pathogenesis of major depressive disorder (MDD), yet whether gut microbiota dysbiosis is associated with the severity of MDD remains unclear. Here, we performed shotgun metagenomic profiling of cross-sectional stool samples from MDD (n = 138) and healthy controls (n = 155). The patients with MDD were divided into three groups according to Hamilton Depression Rating Scale 17 (HAMD-17), including mild (n = 24), moderate (n = 72) and severe (n = 42) individuals, respectively. We found that microbial diversity was closely related to the severity of MDD. Compared to HCs, the abundance of Bacteroides was significantly increased in both moderate and severe MDD, while Ruminococcus and Eubacterium depleted mainly in severe group. In addition, we identified 99 bacteria species specific to severity of depression. Furthermore, a panel of microbiota marker comprising of 37 bacteria species enabled to effectively distinguish MDD patients with different severity. Together, we identified different perturbation patterns of gut microbiota in mild-to-severe depression, and identified potential diagnostic and therapeutic targets.