The objective of this study was to determine effects of replacing soybean meal by urea and negative energy balance (NEB) on nitrogen balance, ruminal fermentation characteristics and the kinetics of plasma glucose and urea in sheep. A crossover design with two different types of isonitrogenous (crude protein 5 g/ (kg^0.75·day) source, either soybean meal or replaced with urea, was assigned to six sheep. High energy dietary treatment (669 kJ/ (kg^0.75·day) following with low energy dietary treatment (222 kJ/ (kg^0.75·day) was nested in each isonitrogenous source for NEB induction. Plasma glucose and urea turnover rates were determined by the primed continuous infusion of [U-¹³C] glucose and [¹⁵N₂] urea simultaneously. NEB induction was modest with indicated by increased (P<0.01) of serum non-esterified fatty acid concentration, while β-hydroxybutyric acid concentration did not change. Nitrogen retention was lower (P<0.01) in NEB. Rumen pH and ammonia concentration were higher with urea replacement (P=0.04) and NEB (P<0.01). Almost all rumen volatile fatty acids concentration and plasma free amino acid concentrations were lower (P<0.01 and P<0.05) in NEB. Plasma glucose and ammonia concentrations were not influenced by urea replacement and NEB. The glucose turnover rate was lower (P<0.01) in NEB. However, plasma urea concentration and turnover rate were higher (P<0.01) in NEB. In conclusion, ruminal fermentation characteristics are affected by urea replacement and NEB. Regardless of urea replacement effect and interaction of urea × energy, NEB itself has strong influence on both of glucose and urea kinetics.