The use of biomarkers for feed conversion efficiency (FCE), such as Nitrogen isotopic discrimination (15N), facilitates easier measurement and may be useful in breeding strategies. However, we need to better understand the relationship between FCE and 15N, particularly the effects of differences in the composition of liveweight gain and rumen N metabolism. Alongside measurements of FCE and 15N, we estimated changes in body composition and used dietary treatments with and without nitrates, and rumen metagenomics to explore these effects. Nitrate fed steers had reduced FCE and higher 15N in plasma compared to steers offered non-nitrate containing diets. The negative relationship between FCE and 15N was strengthened with the inclusion of fat depth change at the 3rd lumbar vertebrae, but not with average daily gain. We identified 1,700 microbial genes with a relative abundance >0.01% of which, 26 were associated with 15N. These genes explained 69% of variation in 15N and showed clustering in two distinct functional networks. However, there was no clear relationship between their relative abundances and 15N, suggesting that rumen microbial genes contribute little to 15N. Conversely, we show that changes in the composition of gain (fat accretion) provide additional strength to the relationship between FCE and 15N.