Nitrogen (N) remobilization from reserves to sinks is essential for seedling establishment and seed production. Cytosolic glutamine synthetase (GS1) is up-regulated during both seed germination and seed filling in plants. However, the specific roles of the individual GS1 isogenes with respect to N remobilization, early seedling vigour, and final seed productivity are not known. In this study, impairment of seed germination and seedling establishment is demonstrated in the single knockout mutant gln1;2, and the double knockout mutant gln1;1:gln1;2. The negative effect of Gln1;2 deficiency was associated with reduced N remobilization from the cotyledons and could be fully alleviated by exogenous N supply. Following reproductive growth, both the single and double Gln1;2-knockout mutants showed decreased seed yield due to fewer siliques, less seeds per silique, and lower dry weight per seed. The gln1;1 single mutant had normal seed yield structure but primary root development during seed germination was reduced in the presence of external N. Gln1;2 promoter-green fluorescent protein constructs showed that Gln1;2 localizes to the vascular cells of roots, petals, and stamens. It is concluded that Gln1;2 plays an important role in N remobilization for both seedling establishment and seed production in Arabidopsis.