Abstract Osteoarthritis (OA) is a degenerative joint disease affecting both humans and animals, particularly older dogs. In recent years, there has been increasing interest in natural therapies that can provide joint protection and reduce the progression of OA. The natural compound, Tabebuia particularly Tabebuia avellanedae (TA), is rich in bioactive compounds including lapachol and flavonoids. The anti-inflammatory and antioxidant properties of TA can slow down cartilage degradation and reduce joint inflammation. To date, there have been very limited studies on dietary supplementation of Tabebuia bark extract in dogs. Thus, this study investigated the beneficial effects of Tabebuia bark extract on joint protection and the prevention of osteoarthritis in beagle dogs. A total of 4 beagles with an initial body weight of 4.64 ± 0.11 kg were assigned to one of two dietary treatments: CON, Basal diet; TRT, CON + 180mg Tabebuia bark extract (2 times per day) in a 2x2 repeated Latin square design for 10 weeks. There was wash-out period for two weeks. The test feed was prepared by mixing top-dressing and water was provided ad libitum. The growth performance (average daily feed intake, daily gain, and body condition score) was measured at initial, week 4, 6, and 10. The fecal and serum samples were collected at week 4 and 10 for 16S rRNA and GC/MS-TOF analysis, respectively. All data were processed using the GLM procedure of SAS. No significant differences in productivity were observed between treatments (P > 0.05). Metabolic pathway analysis using the KEGG database revealed significant alterations in sphinganine, dihydroceramide, and octadecanoic acid (stearic acid), which are associated with joint health. Significant changes were observed in sphinganine and dihydroceramide levels, while octadecanoic acid showed differences in fatty acid synthesis pathways.Gut microbiome analysis showed that Romboutsia, Fusobacterium, Lachnospiraceae, and Megamonas funiformis were more abundant in the control group, whereas the treatment group exhibited reduced levels of these species. The reduction of these microorganisms in the TRT group may contribute to improved health outcomes, including alleviation of inflammatory responses, maintenance of intestinal balance, improvement of metabolic health, and suppression of pathogens. These findings suggest that Tabebuia bark extract could serve as a beneficial supplement in dogs, potentially promoting gut health through microbiome modulation and supporting joint health.