Alternate partial root-zone N-fertigation can improve nitrogen (N) nutrition of barley grown at elevated carbon dioxide (e[CO2]), and the carbon (C) and N metabolism in grain crops may lead to changes in grain quality. However, the effect of N-fertigation under water deficit and e[CO2] on barley grain quality is still largely elusive, and the role of endogenous abscisic acid (ABA) level in grain quality formation under combined drought stress and e[CO2] needs to be further explored. Wild-type barley Steptoe (WT) and its correspondent ABA-deficient barley mutant Az34 were grown at ambient CO2 (a[CO2], 400 ppm) and e[CO2] (800 ppm), respectively. Three N-fertigation regimes, including N-fertigation at full irrigation volume (FIN), N-fertigation at deficit irrigation volume (DIN), and alternate N-fertigation at reduced irrigation volume (PRDN), were imposed from tillering to grain filling stage. When analyzed across the N-fertigation regimes and genotypes, e[CO2] decreased the shoot nitrogen, β-glucan and starch concentrations by 8.3%, 3.6%, and 2.6%, respectively, but increased the grain nitrogen concentration by 6.6% compared to a[CO2]. Az34 had 8.4% and 15.7% higher sulfur and zinc concentrations while 14.3% lower calcium concentration than WT. Compared to FIN, DIN decreased the concentrations of grain nitrogen, phosphorus, sulfur, serine, glutamic acid, proline, threonine, methionine, and leucine by 5.7%, 3.1%, 5.2%, 4.9%, 5.9%, 7.3%, 4.8%, 5.9%, and 5% while increased β-glucan and starch concentrations by 2.2% and 2.9%, respectively; PRDN reduced grain nitrogen, phosphorus, sulfur, serine, glutamic acid, proline, threonine, methionine, and leucine concentrations by 9.4%, 5.1%, 8.2%, 6.4%, 7.6%, 9.5%, 6.5%, 10.9%, and 6.6% while enhanced β-glucan and starch concentrations by 5.3% and 3.8%, respectively. The results provide some novel knowledge about the effect of N-fertigation under water deficit on barley grain quality at e[CO2].