This study investigated the effects of high glucose levels on physiological function and lipid accumulation of Caenorhabditis elegans (C. elegans). C. elegans was incubated with 100 mM glucose for 72 h. Physiological function, fat content and metabolic enzyme expression were tested to determine the effects of high glucose exposure. As a result, C. elegans treated with 100 mM glucose showed shorter body length and decreased production of offspring compared with the worms exposed to normal diet, while no changes in pumping or locomotion were observed. Oil-Red-O (ORO) staining showed increased fat content in C. elegans exposed to high glucose concentration, which suggests defective energy utilization. Considering the importance of enzymes in energy expenditure, the gene expression of 44 enzymes involved in glucose and lipid metabolic pathways were tested using quantitative real-time polymerase chain reaction (qRT-PCR). The results showed that high-glucose treatment affected the gene expression of 10 glucolipid enzymes. A dramatic decline in three enzymes (acs-15, acs-18, acdh-8) involved in mitochondrial beta-oxidation of fatty acids suggests inhibition of fat breakdown. In conclusion, high glucose concentrations induce physiological defects in C. elegans, which might be attributed to impaired fatty acid metabolism in mitochondria.