Objetive： To investigate the mechanism of antidepressant effect of verbascoside based on high-throughput sequencing technology （RNA-Seq），and to explore the possible targets and signaling pathways. MethodsForty C57BL/6 mice were randomly divided into control group，model group，fluoxetine group and verbascoside group，10 mice in each group. Except for the control group，all the other three groups were constructed with chronic unpredictable mild stimulation （CUMS） combined with solitary feeding for four weeks. Control group，fluoxetine group and verbascoside group were administered by gavage once daily for three weeks during the second week of modeling. The mice were assessed by sugar-water preference test，forced swimming test，open field test，elevated cross maze test，and water maze test. Enzyme linked immunosorbent assay（ELISA） was performed to detect the levels of major neurotransmitters and inflammatory factors in mice serum，and mRNA high-throughput sequencing was performed in the nucleus accumben and colon to screen differentially expressed genes and perform pathway enrichment analysis. Real-time polymerase chain reaction（Real-time PCR） was used to detect the mRNA expression of Gad，Slc32a1（VGAT） and brain-derived neurotrophic factor (BDNF） in nucleus accumbens. ResultCompared with the control group，the anxiety and depression-like behaviors in the model group increased，while the learning and memory ability decreased significantly. The content of neurotransmitter in serum decreased significantly. The content of pro-inflammatory factors increased significantly. The mRNA expression of Gad and Slc32a1（VGAT） in nucleus accumbens decreased significantly，while that of BDNF increased significantly. Compared with the model group，the anxiety and depression-like behavior of mice in verbascoside group was significantly relieved. The neurotransmitter content increased significantly，and the pro-inflammatory factors decreased significantly. The mRNA expression of Gad and Slc32a1（VGAT） in nucleus accumbens increased significantly，while that of BDNF decreased significantly.A total of 48 differentially expressed genes in nucleus accumbens and 43 differentially expressed genes in colon were screened by high-throughput sequencing. Differential genes in nucleus accumbens mainly focus on neuroactive ligand-receptor interaction， γ-aminobutyric acid（GABA）ergic synapse，synaptic vesicle cycle and other pathways. Colonic differential genes are mainly concentrated in GABAergic synapses，synaptic vesicle circulation，cyclic adenosine monophosphate（cAMP） signal pathway and other signal pathways. Compared with the control group，the mRNA expression of Gad and Slc32a1（VGAT） in nucleus accumbens of model group decreased significantly，while the mRNA expression of BDNF increased significantly. ConclusionVerbascoside has significant antidepressant effects. Its antidepressant effect may be related to the increase of monoamine neurotransmitters，the decrease of pro-inflammatory factors and the restoration of neurotransmitter homeostasis by increasing GABA，and it mainly acts through the signaling pathways such as neuroactive ligand-receptor interaction，GABAergic synapses，synaptic vesicle cycle and cAMP signaling pathway.