The objective of this study was to assess the relationship between milk urea nitrogen (MUN, mg/dL) and urinary nitrogen (UN) excretion (UN, g/day) in lactating Chinese Holstein cows. Twelve mature lactating cows were divided into four groups by their days in milk and milk yield and fed four different crude protein levels diets (13.2, 14.1, 15.0 and 16.2%, dry matter (DM) basis) with a similar net energy content. The experiment was conducted according to a multiple 4 × 4 Latin square design. The entire length of the experimental periods was 56 days, each period consisting of 14 days with days 1–9 for adjustment and days 10–14 for the collection of urine and data. The crude protein content had significant effects on the MUN content and UN excretion (P < 0.01), but little effect on milk yield and composition (P > 0.05). There was a close relationship between MUN and UN excretion (P < 0.001). The following equations were established to predict UN (g/day) excretion based on MUN (mg/dL) in the lactating Chinese Holstein cows: UN = 10.07 × MUN + 47.29. The results obtained in this study suggested that MUN could be used as a parameter to estimate UN excretion, while the equations to predict UN based on MUN might be somewhat different due to the differences in breeds of cow.