The use of biomarkers for feed conversion efficiency (FCE), such as Nitrogen isotopic discrimination (Delta N-15), facilitates easier measurement and may be useful in breeding strategies. However, we need to better understand the relationship between FCE and Delta N-15, particularly the effects of differences in the composition of liveweight gain and rumen N metabolism. Alongside measurements of FCE and Delta N-15, 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 Delta N-15 in plasma compared to steers offered non-nitrate containing diets. The negative relationship between FCE and Delta N-15 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 Delta N-15. These genes explained 69% of variation in Delta N-15 and showed clustering in two distinct functional networks. However, there was no clear relationship between their relative abundances and Delta N-15, suggesting that rumen microbial genes contribute little to Delta N-15. Conversely, we show that changes in the composition of gain (fat accretion) provide additional strength to the relationship between FCE and Delta N-15.