Elevated CO2 (eCO(2)) has the potential to increase plant biomass while decreasing water demand due to enhanced water-use efficiency (WUE), which interacts with nutritional status. Carbon isotope discrimination (Delta C-13) has been shown to be a valid proxy for estimating WUE; however, its validity is uncertain for plants in an environment where the interaction between CO2 and nutrition strongly affects WUE. Using a single potato cultivar (Irish Cobbler), we examined its validity through three independent trials with varying levels of P, N, or K (Trial P, N, and K, respectively) in growth chambers at two CO2 concentrations. WUE at the plant level varied with CO2 conditions and nutrient supply rates. Plant biomass was positively regressed against WUE in Trials P and K, and water use in Trial N. WUE was negatively regressed against Delta C-13 across various nutrient supply rates within each CO2 environment. However, the relationship between WUE and Delta C-13 was altered with CO2 enrichment by elevating the intercept along the y-axis (WUE) without affecting the slope, implying the involvement of isotopic discrimination in respiration or photorespiration. These results suggest that Delta C-13 can be used to estimate WUE across various nutrient statuses, not only at the current CO2 but also at eCO(2) when the comparisons are made within each CO2 condition.