Nitrogen (N) excretion by cattle, particularly urinary N, can have detrimental environmental impacts on air and water quality, and contributes to greenhouse gas emissions. The effects of increasing the application rate of inorganic N fertiliser from 15 (N15) to 80 (N80) kg/ha per cut to Lolium perenne dominant swards in summer, on intake, rumen fermentation, rumen microbial populations, apparent total-tract digestibility and N-balance in beef cattle, and in vitro fermentation and methane output, were studied. Sixteen suckler-bred Charolais steers, used in a randomised block design experiment, were offered fresh grass mechanically harvested 21-d after N fertiliser application. Similar grass was incubated in an eight-vessel in vitro RUSITEC system. Grass crude protein concentration was 50 g/kg dry matter (DM) higher for N80 compared to N15. There was no difference in grass DM intake between treatments. Rumen fermentation variables did not differ between treatments, except for the molar proportion of propionate, which was greater for N80 than N15. Gross microbial community structure in the rumen was not significantly altered by inorganic N fertiliser application rate. The relative abundance of individual genera Lachnospiraceae_NK3A320_group and Ruminococcaceae_NK4A214_group, and Acetitomaculum were significantly lower at the higher N application rate. Mean plasma urea concentration was greater for N80 compared to N15. In vivo digestibility of DM, organic matter, neutral detergent fibre and acid detergent fibre was unaffected by fertiliser N application rate. Nitrogen intake was 75 g/d greater, and urinary and faecal N excretion were 20 and 5 g/d greater, respectively, for N80 than N15. The quantity of N retained and N use efficiency was greater for N80 compared to N15. In vitro NH3 concentration was greater for N80 than N15, whereas other rumen fermentation variables, and in vitro methane and total gas output, did not differ between treatments. Reducing the inorganic N fertiliser rate applied to mid-season temperate grass reduced N excretion from beef cattle, which is environmentally beneficial, with no effect on in vitro methane production.