Background & Objective: Obesity is a major public health problem whose prevalence has constantly increased worldwide. Obesity is a risk factor for several diseases such as type 2 diabetes, fatty liver disease, hyperlipidemia, cardiovascular diseases, and cancer, all of which reduce the quality of life and life expectancy. This study investigated anti-adipogenic effects of Eryngium billardierei (Ery) extract. Materials & Methods: The nontoxic concentration of Ery extract was estimated via the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazoliu bromide (MTT) assay. The differentiation of mouse-derived 3T3-L1 preadipocytes into mature adipocytes was induced in the presence or absence of Ery extract. Then, the accumulation of lipid droplets was identified by Oil Red O staining. Triacylglycerol level and glycerol3phosphate dehydrogenase (GPDH) activity were also measured. Results: The MTT assay showed that 3 mg/mL of the Ery extract can be a nontoxic dose for other analyses. Oil Red O staining demonstrated a reduction of lipid droplets in the extract-treated adipocyte 3T3-L1 compared to the non-treated group. A significant decrease of triacylglycerol level in the treated 3T3-L1 adipocyte (0.32±0.03 mgTG/mg protein) compared with the nontreated group (0.78±0.05 mgTG/mg protein) (P<0.01), and a significant decrease of glycerol-3-phosphate dehydrogenase (GPDH) activity in the 3T3-L1adipocyte treated (15.3±2.1 U/ml) compared to the non-treated group (30.2±3.9 U/ml) (P<0.05) confirmed the inhibitory action of Ery extract on adipocyte differentiation. Conclusion: Ery extract, as a safe herbal extract, has anti-adipogenic activity and merits more investigation as a candidate for developing an anti-obesity drug or supplement.