Dairy cows experience an increased demand for glucose to support milk production. However, negative energy balance is a common condition in peripartum cows. In response, fat mobilization provides non-esterified fatty acids (NEFAs) for oxidation in the liver to generate ATP. To investigate the effects of NEFAs on gluconeogenesis, the expression and enzyme activity of pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPCK) in cultured bovine hepatocytes were evaluated by quantitative polymerase chain reaction and spectrophotometry, respectively. The results showed that PC and PEPCK mRNA levels were marked decreased when the NEFAs concentrations exceeded 0.5 and 1.5 mmol/l, respectively. The PC and PEPCK enzyme activity showed significantly decreased when the NEFAs concentrations exceeded 1.5 and 0.5 mmol/l, respectively. These findings indicate that high circulating levels of NEFAs inhibit hepatocyte gluconeogenesis, thereby promoting negative energy balance.