The gut microbiota is considered the host's "second genome" and is closely associated with the host's physiology. We found that the elimination of gut bacteria suppressed ovarian development in Bactrocera dorsalis. Proteomic analysis revealed significant differences in ovarian protein expression after gut bacteria depletion, with differentially expressed proteins enriched in the proteasome and ubiquitin-related pathway. Moreover, ubiquitination levels were significantly reduced in gut bacteria-depleted females, while Enterobacter hormaechei (EH) supplementation rescued ubiquitination levels. Our findings suggest that ubiquitination serves as a mediator through which gut bacteria regulate ovarian development. By ubiquitin-modified proteomic analysis following gut microbiota manipulation and EH supplementation, we aimed to identify ubiquitination targets critical for ovarian development.