Environmental metabolomics has become interesting in marine ecological studies. One example is the revealing of new insights in stress response of Zostera marina. This is essential to understand how, at which level and to what extend aquatic plants adapt, tolerate and react to environmental stressors. We exposed Z. marina to water column anoxia and assessed the diurnal metabolomic response by GC-TOF-MS based metabolomics identifying 109 known and 217 unknown metabolites. During day time photosynthetic oxygen production prevents severe effects of anoxia on the metabolome (complete set of small-molecule metabolites). Night time water column anoxia caused a shift in metabolic composition and concentration (trajectories of PLS-DA) in particular an increase in pyruvate and lactate indicating an activation of fermentation in the plant. Concurrent increase of alanine, succinate and GABA suggests the presence of the GABA shunt pathway, a strategy to decrease pyruvate levels by transamination. The down regulation of TCA cycle intermediates illustrates the inactivity of TCA due to lack of oxygen, which results in an accumulation of pyruvate and followed by fermentation to lactic acid. We demonstrate the applicability of metabolomics to assess environmental stress responses of Zostera marina.