The aim of the study was to investigate the impact of lameness on nitrogen (N) metabolism and excretion. Two treatment groups of 20 multiparous Holstein–Friesian dairy cows were included in study; the control group consisted of cows with locomotion score ≤2, while the experimental group consisted of cows with locomotion scores 3 and 4. Fodder, milk, feces, and urine were collected to determine nitrogen emissions. The milk yield, the energy-corrected milk, the fat and protein yield were higher for lame animals compare with non-lame cows. Differences were also detected in the milk urea nitrogen (MUN) between groups where lame cows had a 15% lower MUN than non-lame animals. Urine volume was lower (p < 0.008), while urinary creatinine concentration was higher (p < 0.05) in lame animals compare with those in the non-lame group. Consequently, the creatinine/urea ratio was significantly higher (p < 0.001) in the lame vs. no-lame animals. Nitrogen excretion in milk was higher (p = 0.008) and N in urine was lower (p < 0.001) in the lame compared to non-lame cows with lower urinary N emissions in lame animals. Taken together, our results show that urinary creatinine concentration and urinary creatinine/urea ratio have the potential to be used as a tool for lameness detection.