Milk urea concentration is an indicator for dietary nitrogen (N)-supply and urinary nitrogen excretion. Dairy cows with high (HMU) compared to low milk urea (LMU) concentration have greater plasma urea, creatinine and uric acid concentrations, but if the liver metabolism accounts for these differences is unknown. Eighteen HMU and 18 LMU cows were fed either a diet with a low (LP) and normal (NP) crude protein concentration. An N-balance study was performed and a 13C-urea bolus was administered followed by a series of blood samplings. Liver samples were analysed by 2D-gel-based proteomics and real-time RT-PCR. Although HMU cows had a higher urea pool, plasma urea, uric acid, and hippuric acid concentrations, these differences were not associated with increased activation of the hepatic urea cycle or non-urea nitrogen metabolite pathways. Instead, HMU cows had a higher oxidative stress level. Conclusively, other factors than hepatic urea metabolism account for the milk urea concentration. Despite higher plasma urea concentrations on the LP diet, urea cycle mRNA expression was not affected, indicating it is not controlled at transcriptional level. Feeding the LP diet resulted in higher expressions of enzymes catabolizing fatty acids, which is likely due to diminished microbial growth and insufficient energy supply.