Two consecutive trials were conducted to investigate the effects of glucosinolates (GLS) in rapeseed cake (RSC) on nitrogen (N) metabolism and urine nitrous oxide (N
2
O) emissions in steers. In trial 1, 8 steers and 4 levels of RSC, i.e. 0, 2.7%, 5.4% and 8.0% dry matter (DM) (0, 6.0, 12.1, 18.1 μmol GLS/g DM) were allocated in a replicated 4 × 4 Latin square. In trial 2, the static incubation technique was used for measuring the N
2
O emissions of the urine samples collected from trial 1. The results of trial 1 indicated that dietary inclusion of RSC decreased the digested N and increased the fecal N excretion (
P
< 0.01), whereas it did not affect the urinary N excretion, total N excretion and N retention (
P
> 0.10). Dietary inclusion of RSC decreased the urinary excretion of urea while it increased allantoin, total purine derivatives, the predicted rumen microbial N flow and thiocyanate (SCN) (
P
< 0.05). Dietary inclusion of RSC did not affect the plasma triiodothyronine and thyroxine while it down-regulated the plasma relative concentrations of 4-aminohippuric acid, 3α,7α-dihydroxycoprostanic acid, phosphatidylserine (14:0/16:0), 6β-hydroxyprogesterone, pyrrhoxanthinol, tatridin B, mandelonitrile rutinoside, taraxacoside (
P
< 0.05), and up-regulated hypoglycin B, neuromedin N (1-4), dhurrin, 5-deoxykievitone (
P
< 0.01). The results of trial 2 indicated that dietary RSC increased the steer urine N
2
O–N fluxes, the ratio of N
2
O–N to N application and the estimated steer urine N
2
O–N emissions (
P
< 0.01). A close correlation was found between the estimated steer urine N
2
O–N emissions and the output of urinary SCN (
P
< 0.001). In conclusion, dietary RSC increased the fecal N excretion, whereas it did not affect the urinary N excretion and the N retention rate in steers. Dietary RSC increased rather than decreased the urine N
2
O–N emissions even though it decreased the urinary excretion of urea. The SCN excreted in urine could be the major factor in increasing the urine N
2
O–N emissions. Whether other metabolites excreted into urine from RSC have an impact on the urine N
2
O–N emissions in steers needs to be investigated in the future.