Background and Aims <p>This study aimed to elucidate the mechanisms of root-based inorganic carbon (RIC) uptake and fixation in <i>Arabidopsis thaliana</i> and barley (<i>Hordeum vulgare</i> ‘Golden Promise’) and compare the physiological response of the two species under osmotic stress.</p> Methods <p>Barley plants were grown in a growth-promoting inorganic carbon (IC)-containing hydroponic solution. Analyses included fluxomics, physiological measurements, amino acid profiling in leaves, and RNA sequencing. PEG-induced osmotic stress assays were performed for barley and Arabidopsis. Fixation of ¹³C-labelled carbon into shoot dry mass was studied for <i>Arabidopsis thaliana</i> genotypes Col and high suberin-producing mutants <i>sultr1;2</i> and <i>esb1-1.</i></p> Results <p>IC treatment improved barley growth and net photosynthesis, though the effects were less pronounced than in Arabidopsis. In barley, IC fixation occurred mainly in roots via phosphoenolpyruvate carboxylase (PEPC), supported by fluxomic and transcriptome data. The high suberin-producing Arabidopsis mutants took up and fixed less ¹³C from the hydroponic solution, but no change of suberin-related pathways was observed in the transcriptome of IC-treated barley. Amino acid profiling and transcriptomics in barley leaves revealed an increased use of pyruvate-derived amino acids (leucine, alanine, valine, and isoleucine), supporting protein synthesis and photosynthesis. In the leaves, genes related to cell division and cell expansion, and chloroplast biosynthesis were upregulated. Under PEG-induced osmotic stress, IC-supplementation improved the growth of Arabidopsis and some photosynthetic parameters of barley.</p> Conclusions <p>The observed growth promotion in barley under root-IC treatment is driven by carbon uptake and fixation pathways that differ significantly from the mechanisms utilized by Arabidopsis.</p>