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Background: Negative energy balance (NEB) is a common pathological foundation of ketosis and fatty liver. Liver and fat tissue are the major organs of lipid metabolism and take part in modulating lipid oxidative capacity and energy demands, which is also a key metabolic pathway that regulates NEB develop during perinatal period. Fibroblast growth factor-21 (FGF-21) is a recently discovered protein hormone that plays an important and specific regulating role in adipose lipid metabolism and liver gluconeogenesis for human and mouse. Our aim is to investigate the variation and relationship between serum FGF-21 concentration and characteristic parameters related to negative energy balance in different energy metabolism state., Methods: In this research, five non-pregnant, non-lactating Holstein-Friesian dairy cows were randomly allocated into two groups. The interventions were a controlled-energy diet (30% of maintenance energy requirements) and a moderate-energy diet (120% of predicted energy requirements) that lasted for the duration of the experiment. We measured biochemical parameters, serum FGF-21, leptin and insulin levels by commercial ELISA kits., Results: The results showed that serum FGF-21 levels were significantly higher in both groups treated with a controlled-energy diet, while FGF-21 levels in both groups treated with moderate-energy diet were low. FGF-21 levels exhibited a significant positive correlation with serum leptin levels, while an inverse relationship was found between FGF-21 and blood glucose and β-hydroxybutyrate acid (BHBA) levels., Conclusion: An increase in FGF-21 levels after a controlled-energy diet treatment may contribute to a positive metabolic effect which could result in a new theoretical and practical basis for the preventive strategy of dairy cows with NEB.