The aim of this study was to investigate whether a water extract of L. cladonioides (LC) has an anti-obesity effect in 3T3-L1 cells and obese mice. Treatment of differentiated 3T3-L1 adipocytes with LC caused a significant increase in glycerol release and reduced the protein expression of the adipogenic transcription factors, PPARγ and C/EBPα. In an animal model, obese mice were artificially induced by a high fat diet for 10 weeks. Experimental groups were treated with LC (100 mg/kg/day) by gavage for the next 10 weeks. At the end of experiment, the body weight of the LC group mice was reduced by 14.2% compared to the high fat diet (HFD) group. The treatment also decreased liver (31.0%), epididymal (18.0%) and retroperitoneal (19.3%) adipose tissue, and kidney (6.7%) weights, respectively, compared with those of the HFD group. LC prevented diet-induced increases in the serum level of TC (22.6%), TG (11.6%), and glucose (35.0%), respectively, compared with the HFD group. However, the HDL-C level was higher in the LC group (26.1%) than the HFD group. The results of this study thus suggest that LC suppressed lipid accumulation and expression of adipogenic transcription factors, and increased the amount of glycerol release. LC also indicated an anti-obese and anti-hyperlipidemic effect.