The aging of our population has become one of the major concerns in Taiwan. Sarcopenia, the degenerative loss of skeletal muscle mass, quality and strength associated with aging, is one of the most important causes of functional decline and loss of independence in the elderly. Maintaining the mass and functions of skeletal muscles is hence a key issue for healthy aging. Estrogen-related receptor α, ERRα, is required for the activation of mitochondrial genes, and known to regulate genes of mitochondrial biogenesis and energy metabolism. The essential role of ERRα in skeletal muscle fitness and regeneration has been demonstrated. Previously, wild bitter gourds (WBG) were found to elevate mitochondrial biogenesis markers in skeletal muscles in mice. As some active compounds in WBG shows some structural similarity to the endogenous ligand of ERRα, we hypothesize that WBG can promote mitochondrial activity by regulating ERRα. A cell-based ERRα transactivation assay and an animal experiment were conducted to test this hypothesis. ERRα ligand binding domain was first cloned and then used to develop the cell-based transactivation assay. In this assay, XCT790, a synthetic inverse agonist, activated ERRα at low concentrations, but showed antagonism at higher ones. Genistein, a reported ERRα ligand, dose-dependently inhibited transactivation. A total of five samples, including four cultivars, 1758, P81, 55M and H4, and the product of P81 hydrolyzed at 50℃, P81_50, were tested in this assay. The ethyl acetate extracts of the WBGs also showed dose-dependently inhibitions which were to a greater extent than that of genistein. All the five samples exhibited comparable effects. Among the three active constituents of WBG, α-eleostearic acid, a conjugated linolenic acid showed the highest activity. In the mice study, groups of C57BL/6J mice were fed the high fat diet supplemented with 4% of lyophilized WBG powder for four weeks. The mRNA gene expressions of ERRα and its downstream genes in the skeletal muscles were analyzed. Among these, the mitochondrial dynamics marker, mitofusin 2, which contains ERRα response element within its promoter, was significantly up-regulated in the soleus muscle of mice fed the P81 cultivar of WBG. In conclusion, this study provided preliminary evidence that constituents of WBG might regulate ERRα activity, and promote mitofusin 2 mRNA expression in soleus muscle of high-fat diet fed mice. These results suggest that WBG might have health benefit through the modulation of ERRα.