The metabolic profiles of rice and barley plants constitutively expressing a sodium-pumping ATPase (PpENA1) isolated from the bryophyte Physcomitrella patens were examined using GC-MS. Quantitative real-time PCR (qRT-PCR) was used to determine the mRNA levels of PpENA1 in root and leaf tissues of the transgenic rice and barley lines. PpENA1 mRNA levels were significantly higher in rice lines than in barley lines with the same dual CaMV35S promoter controlling PpENA1 transcription in both species. In rice, PpENA1 mRNA levels were greatest in the shoot whilst levels were greatest in the roots of barley. Metabolite profiles were determined in the flag leaf of both rice and barley plants grown under controlled conditions. A large proportion of the measured metabolites were significantly altered in the transgenic lines compared to null-segregating lines, revealing a considerable impact of expression of the sodium-pumping ATPase (PpENA1) transgene on metabolism. Interestingly, the metabolite changes were different between rice and barley, indicating different responses of rice and barley to the introduction of this gene.