Nitrogen (N) is an essential macronutrient, and the final form of endogenous inorganic N is ammonium, which is assimilated by Gln synthetase (GS) into Gln. However, how the multiple isoforms of cytosolic GSs contribute to metabolic systems via the regulation of ammonium assimilation remains unclear. In this study, we compared the effects of two rice (Oryza sativa) cytosolic GSs, namely OsGS1;1 and OsGS1;2, on central metabolism in roots using reverse genetics, metabolomic and transcriptomic profiling, and network analyses. We observed (1) abnormal sugar and organic N accumulation and (2) significant up-regulation of genes associated with photosynthesis and chlorophyll biosynthesis in the roots of Osgs1;1 but not Osgs1;2 knockout mutants. Network analysis of the Osgs1;1 mutant suggested that metabolism of Gln was coordinated with the metabolic modules of sugar metabolism, tricarboxylic acid cycle, and carbon fixation. Transcript profiling of Osgs1;1 mutant roots revealed that expression of the rice sigma-factor (OsSIG) genes in the mutants were transiently upregulated. GOLDEN2-LIKE transcription factor-encoding genes, which are involved in chloroplast biogenesis in rice, could not compensate for the lack of OsSIGs in the Osgs1;1 mutant. Microscopic analysis revealed mature chloroplast development in Osgs1;1 roots but not in the roots of Osgs1;2, Osgs1;2-complemented lines, or the wild type. Thus, organic N assimilated by OsGS1;1 affects a broad range of metabolites and transcripts involved in maintaining metabolic homeostasis and plastid development in rice roots, whereas OsGS1;2 has a more specific role, affecting mainly amino acid homeostasis but not carbon metabolism.