Climate change is shifting upward the elevational limit of many species. When plants migrate along elevational gradients to follow their thermal niche, they experience a decrease in atmospheric pressure the higher they migrate. The direct effect of low air pressure on plant morphology, physiology, and metabolism remains unclear. Using Ecotron chambers that independently control atmospheric pressure and other climatic drivers, we studied a broad spectrum of physiological and metabolic parameters of the model plant Arabidopsis thaliana at two different air pressures (101 and 62 kPa). We found a decrease in stomatal conductance, CO2 assimilation rate, and internal-to-atmospheric CO2 ratio at lower atmospheric pressure. Interestingly, despite reduced gas exchange, we observed an increase in leaf 13C abundance, suggesting enhanced carboxylation capacity. Moreover, at 62 kPa, we observed a higher leaf accumulation of secondary metabolites, a defense mechanism that plants usually adopt under stress conditions. Our results confirm that air pressure is a key abiotic factor influencing plant performance and fitness across a broad spectrum by modifying both physiological and metabolic processes. Therefore, understanding the mechanisms behind a model plant’s response to low air pressure is essential for incorporating this factor into future studies on plant migration along elevational gradients.