A complex interplay of environmental factors profoundly influences plant cellular metabolism, with gaseous exchange serving as a fundamental physiological process critical to growth and survival. While well-characterized in natural environments, the role of gaseous exchange in plant in-vitro culture remains underexplored. Plant in-vitro culture offers a versatile platform for studying metabolism, where metabolic networks are highly flexible and sensitive to environmental factors. Despite advancements in understanding these dynamics, there has been relatively little investigation into how gaseous exchange within tissue culture systems affects cellular metabolism. In the present study, we investigated the effects of gaseous exchange on plant growth and metabolism by comparing traditional Parafilm- and micropore-tape-based cultures designed to facilitate different levels of gaseous exchange. A comprehensive metabolomics approach using liquid chromatography-high-resolution mass spectrometry and gas chromatography-mass spectrometry was employed to delineate the metabolic changes in Arabidopsis under Parafilm- and micropore-tape-sealed culture conditions at two and three weeks of growth. Metabolic profiling identified increased levels of oxidized glutathione, arginine, ornithine, and aspartic acid, and decreased levels of TCA cycle intermediates and phenylpropanoid metabolites, indicating that restricted gas exchange alters the redox status and reprograms primary and secondary metabolism. This reprogramming affected amino acid metabolism, arginine metabolism, energy metabolism, as well as phenylpropanoid and flavonoid biosynthetic pathways. Restricted gaseous exchange in Parafilm-wrapped cultures also led to altered accumulation of several essential macro- and microelements in Arabidopsis seedlings. The present study demonstrated that restricted gaseous exchange inhibits plant growth and disrupts metabolism.