Hypertrophy of adipose tissues and dysbiosis are hallmarks of obesity. Although drugs are applied for obesity treatment, side effects limit their use. The anti-obesity capacity of rosmarinic acid (RA) has been documented. Trichodesma khasianum Clarke is an edible RA-rich plant grown in Taiwan. Our previous study found that an 80 % ethanol extract of T. khasianum Clarke leaves (80EETC) ameliorates gastric mucosal damage through its anti-inflammatory, antioxidant, and microbiota modulation abilities. However, the anti-obesity effect of 80EETC remains unclear. Therefore, the objective of this study was to explore the protective effects of low-dose 80EETC (125 mg/kg b.w., 80EETCL) or high-dose 80EETC (250 mg/kg b.w., 80EETCH) on obesity development through gut microbiota modulation in high-fat diet (HFD)-induced C57BL/6 mice. The results showed a high RA content (89.2 ± 7.4 mg/g) in 80EETC. 80EETC administration significantly decreased body weight, body fat ratio, serum lipid levels (TC, TG, and LDL-C), adipose tissue accumulation, malondialdehyde (MDA), and tumor necrosis factor-α (TNF-α) in HFD-fed mice. Furthermore, supplementation with 80EETC reduced the Firmicutes/Bacteroidetes ratio and enhanced the relative abundance of gut microbiota (p_Bacteroidetes, f_Lactobacillus, f_Muribaculaceae, f_Prevotellaceae, g_Lactobacillus, g_Prevotellaceae_NK3B31_group, g_Ruminococcaceae_UCG–013, and g_Ruminococcaceae_UCG–014), which negatively correlated with obesity-related factors such as body weight, energy intake, fat accumulation in adipose tissue, TC, TG, LDL, and MDA. In conclusion, RA-rich 80EETC had a protective effect against obesity development and it has potential in healthy food applications.