AbstractMajor depressive disorder (MDD) is a serious mental illness. Increasing evidence from both animal and human studies suggested that the gut microbiota might be involved in the onset of depression via the gut–brain axis. However, the mechanism in depression remains unclear. To explore the protein changes of the gut–brain axis modulated by gut microbiota, germ-free mice were transplanted with gut microbiota from MDD patients to induce depression-like behaviors. Behavioral tests were performed following fecal microbiota transplantation. A quantitative proteomics approach was used to examine changes in protein expression in the prefrontal cortex (PFC), liver, cecum, and serum. Then differential protein analysis and weighted gene coexpression network analysis were used to identify microbiota-related protein modules. Our results suggested that gut microbiota induced the alteration of protein expression levels in multiple tissues of the gut–brain axis in mice with depression-like phenotype, and these changes of the PFC and liver were model specific compared to chronic stress models. Gene ontology enrichment analysis revealed that the protein changes of the gut–brain axis were involved in a variety of biological functions, including metabolic process and inflammatory response, in which energy metabolism is the core change of the protein network. Our data provide clues for future studies in the gut–brain axis on protein level and deepen the understanding of how gut microbiota cause depression-like behaviors.