Background/ Objective <p>Bone marrow mesenchymal stem cells (BMSCs) contribute to controlling adipose tissue homeostasis and may be a potential target for obesity therapy. Since lack of connexin43(Cx43) in mesenchymal stem cells (MSCs) is known to combat high-fat diet (HFD)-induced adiposity. Also, HFD is a well-known risk factor for gut microbiota dysbiosis. Here we attempt to address the interaction between HFD, gut microbiota, and Cx43 deficiency in BMSCs during the development of obesity.</p> Methods <p>BMSC-specific Cx43 knockout (<i>Prrx1-Cre</i>;Cx43^{<i>flox/flox</i>}(cKO)) and control littermate (Cx43^{<i>flox/flox</i>}(flox))mice were fed a regular chow diet or a HFD for 8 weeks. Cecal microbiome composition was assessed by 16S ribosomal RNA (rRNA) sequencing, and further analyzing correlation between gut microbiota and obesogenic parameters. Moreover, transplantation fecal microbiota was used to probe the salutary effect of BMSC Cx43.</p> Results <p>HFD fed cKO mice notably altered cecal microbiome composition as proved by a distinctively clustered separation from flox mice. Concomitantly, <i>Allobaculum</i> induced by HFD was strongly correlated positively with body weight, glucose intolerance, and adipose mass. The recipients with gut microbiota from cKO mice displayed anti-obesity phenotype as the donors.</p> Conclusions <p>Gut microbiota alteration may be one main reason for the salutary effect of BMSC Cx43 knockout on body adiposity.</p>