Negative energy balance (NEB) is a common pathological foundation of fatty liver and ketosis. Liver and fat tissue are the major organs of lipid metabolism, which is also a key metabolic pathway that regulates NEB development during the perinatal period. Fibroblast growth factor-21 (FGF-21) is a protein hormone that plays an important role in adipose lipid metabolism and liver gluconeogenesis. Our aim was to investigate the effects of exogenous FGF-21 on characteristic parameters related to energy balance in dairy cows. Ten non-pregnant, non-lactating Holstein-Friesian dairy cows were randomly allocated into two groups. The interventions were exogenous FGF-21 injection group received 1 ug/kg BW (body weight) of recombinant bovine FGF-21 by intravenous injection, and control group received physiological saline injections by intravenous injection. In comparison to saline injections, intravenous injections of FGF-21 either increased or tended to increase concentrations of FGF-21 (p < 0.05), BHBA (p < 0.05), adiponectin, leptin and HDL-C. FGF-21 injections decreased or tended to decrease concentrations of insulin, glucose, glucagon (p < 0.05), ALT/GPT (p < 0.05), AST/GOT (p < 0.05), urate, creatinine (p < 0.05), BUN, triglyceride (p < 0.05), T-CHO and LDL-C (p < 0.05). The results indicate that FGF-21 has only negative effects on the metabolites and metabolic hormones related to NEB in serum of dairy cows, but it has more beneficial effects on prominent adipokines, liver function index, renal function index, lipoprotein profiles related to the pathological changes that occurred in NEB.