Many studies suggest that Antrodiacinnamomea (AC) has the potential to prevent or alleviate liver diseases, hypertension, diabetes, inflammatory, tumorigenic diseases,and various cancers.Recently, AC has been shown toprevent metabolic disorders ofdiet-induced obesity and the development offatty liver and regulate gut microbiota. Some studies suggest that AC could be a potential prospective nutraceutical in the treatment of obesity or as a daily remedy in the prevention of disease.However, the AC-inducedeffects on the gut-liver axis and the gut microbial regulation are mostlyunknown; meanwhile, whether the AC protects or attenuates the inherited obesity in leptin-deficient (ob/ob) miceisalso unclear.Therefore, the two aims of this study, the aim I was to investigate the AC-induced effect in C57BL/6 (B6) mice, and the aim II was to understand the anti-obesity effect of AC in ob/ob mice. In the present study, theB6 mice wereused to mimic healthy human and ob/ob miceas an over-feeding animal model.TheB6 mice and ob/ob mice werefed with or without AC for 4 weeks. First, thetwo-dimensional difference gel electrophoresis (2D-DIGE) with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI Biotyper were used to investigate the comprehensive influence of AC in the gut-liver axis and gut microbiota of B6 mice.Next, thereal-time PCR and immunoblotting analysis wereperformed to elaborate theanti-obesity mechanism of AC. Furthermore, we used Caco-2 cells as a human intestinal epithelial barrier model to examine the effect of AC and metabolite of bacteria on intestinal permeability.The results of AC-induced effects in B6 miceshowed thatthe body weight, glucose tolerance test (GTT), daily food/water intake and fecal/urine weight were unaffected.Besides, AC supplementation mightdecrease hepatic lipid content by increasing metabolism and interacting intestinal permeability by increasing redox regulation, cytoskeleton regulation and metabolismin B6 mice. Meanwhile, AC treatment increased the abundance of Alistipesshahii (AS) in the gut microbiota ofB6 mice. The gut microbiota mightinteract with the gut-liver axis mainly thought metabolites of bacteria, therefore, we utilized the metabolite of Alistipesshahii (MAS) to understand the effect on colorectal cancer cellsSW480, high-invasivecolorectal cancer cells SW480-I5 and Caco-2 cell barrier model.The results showed that MASwasnot obviouslycorrelated with cell viability and migration ability on SW480 and SW480-I5, and the MAS might increase intestinal permeability in Caco-2 cells. Next,the results ofAC-inducedanti-obesityability in ob/ob miceindicated that AC supplementation suppressed hepatic lipogenesis and lipid uptake andfacilitated lipolysis and reduces lipogenesis to prevent fat deposition in the epididymal white adipose tissue (EWAT). AC also reinforced intestinal barrier integrity and reduced intestinal permeability as preventive protection in ob/ob mice and the Caco-2 cellbarrier model.Taken together, our studyelaborated that AC-induced mechanisms on gut microbiota alteration and differential protein expression of the gut-liver axisinB6 mice.Simultaneously, AC supplementation effectively reduced leptin-deficiency-mediated obesity by regulating metabolism in the liver and EWAT and restoring the gut barrier integrity without any significant compromise on food intake. Thus, our study provides a comprehensive assessment for AC as the nutraceutical, and the AC extract effectively reduced obesity caused by leptin-deficiency and could potentially be used as a nutraceutical for treating obesity.