The main objective of this study was to investigate the relationship between partitioning and isotopic fractionation of nitrogen (N) in sheep consuming diets with varying ratios of N to water-soluble carbohydrate (WSC). Six non-lactating sheep were offered a constant dry matter (DM) allowance with one of three ratios of dietary N/WSC, achieved by adding sucrose and urea to lucerne pellets. A replicated 3 dietary treatments (Low, Medium and High N/WSC) × 3 (collection periods) and a Latin square design was used, with two sheep assigned to each treatment in each period. Feed, faeces, urine, plasma, wool, muscle and liver samples were collected and analysed for 15N concentration. Nitrogen intake and outputs in faeces and urine were measured for each sheep using 6-day total collections. Blood urea N (BUN) and urinary excretion of purine derivative were also measured. Treatment effects were tested using general ANOVA; the relationships between measured variables were analysed by linear regression. BUN and N intake increased by 46% and 35%, respectively, when N/WSC increased 2.5-fold. However, no indication of change in microbial protein synthesis was detected. Results indicated effects of dietary treatments on urinary N/faecal N, faecal N/N intake and retained N/N intake. In addition, the linear relationships between plasma δ15N and urinary N/N intake and muscle δ15N and retained N/N intake based on individual measurements showed the potential of using N isotopic fractionation as an easy-to-use indicator of N partitioning when N supply exceeds that required to match energy supply in the diet.