Atmospheric concentrations of the greenhouse gas nitrous oxide (N2O) have continued to rise since the advent of the industrial era, largely because of the increase in agricultural land use. The urine deposited by grazing ruminant animals is a major global source of agricultural N2O. With the first commitment period for reducing greenhouse gas emissions under the Kyoto Protocol now underway, mitigation options for ruminant urine N2O emissions are urgently needed. Recent studies showed that increasing the urinary concentration of the minor urine constituent hippuric acid resulted in reduced emissions of N2O from a sandy soil treated with synthetic bovine urine, due to a reduction in denitrification. A similar effect was seen when benzoic acid, a product of hippuric acid hydrolysis, was used. This current laboratory experiment aimed to investigate these effects using real cow urine for the first time. Increased concentrations of hippuric acid or benzoic acid in the urine led to reduction of N2O emissions by 65% (from 17% to <6% N applied), with no difference between the two acid treatments. Ammonia volatilization did not increase significantly with increased hippuric acid or benzoic acid concentrations in the urine applied. Therefore, there was a net reduction in gaseous N loss from the soil with higher urinary concentrations of both hippuric acid and benzoic acid. The results show that elevating hippuric acid in the urine had a marked negative effect on both nitrification and denitrification rates and on subsequent N2O fluxes. This study indicates the potential for developing a novel mitigation strategy based on manipulation of urine composition through ruminant diet.