Obesity significantly impacts human health, making dietary intervention a key research focus. Chrysin, a natural flavonoid with diverse bioactivities, shows potential as an obesity adjuvant therapy. However, its interaction with obesity-related gut microbiota remains unclear. Our study demonstrated that chrysin significantly mitigated overweight, insulin resistance, metabolic dysregulation, and liver damage in obese mice. Moreover, chrysin improved gut microbiota disorders to promote the proliferation of beneficial bacteria such as Bacteroides, Parabacteroides and Blautia. It also elevated the level of microbiota metabolites including indole-3-carboxaldehyde and certain bile acids. Consistently, chrysin increased the concentration of short-chain fatty acids (SCFAs) and enhanced intestinal barrier function. Furthermore, chrysin reduced lipopolysaccharide (LPS), tumor necrosis factor (TNF)-α, and interleukin (IL)-6 levels, and suppressed the expression of Toll like receptor 4 (TLR4), c-Jun N-terminal kinase (JNK) and nuclear factor kappa-B (NF-κB), thereby activating protein kinase B (AKT) and adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK). Notably, fecal microbiota transplantation experiments substantiated the critical role of the gut microbiota in mediating the anti-obesity effects of chrysin. In summary, our research indicates that chrysin alleviated obesity through the modulation of gut microbiota and the TLR4/JNK signaling pathway, thereby establishing a scientific rationale for considering chrysin as a potential therapeutic agent for obesity.