Abstract: In the study, we employed an untargeted metabolomic approach in conjunction with 16S rRNA high-throughput sequencing to identify potential antidepressant active effectors mediated by gut-flora among geniposides. Firstly, the rat depressed model was constructed using chronic unpredictable mild stress stimulation (CUMS) combined with orphaned model. Then the cecal contents of the rats were analyzed by untargeted metabolomics using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q/TOF-MS) in order to identify differential metabolites. The intestinal genera exhibiting significant differences in the cecal contents were identified through 16S rRNA high-throughput sequencing, and correlation analysis was subsequently conducted between the differential metabolites and the intestinal genera. The comparison of the metabolic profiles between the normal control group, the depression model group, and the geniposide treatment group revealed that 147 metabolites were down-regulated and 381 metabolites were up-regulated in the model group compared with the normal group. Furthermore, 212 metabolites were up-regulated and 288 metabolites were down-regulated in the geniposide treatment group compared with the model group. Subsequently, the combination of the screening conditions of FC (fold change) > 2, P < 0.05 and VIP (variable importance in the projection) > 1, secondary mapping and database comparison led to the identification of 55 metabolites with significant differences among the three groups. The results of the metabolic pathway enrichment analysis indicated that the identified differential metabolites were primarily involved in five metabolic pathways, namely tryptophan metabolism, arginine biosynthesis, phenylalanine biosynthesis with tyrosine and tryptophan, phenylalanine metabolism, and arginine metabolism with proline. The 16S rRNA sequencing results further indicated that Gardenia jasminoides extract may exert its antidepressant effects, thereby providing a new basis for the study of the gut-brain axis in the therapeutic strategy of depression. All animal experiments were conducted with the approval of the Biomedical Research Ethics Committee of the Naval Medical University.