Rice (Oryza sativa) glutamate synthase (GOGAT, EC 1.4.1.14) enzymes have been proposed to have great potential for improving nitrogen use efficiency, but their functions in vivo and their effects on carbon and nitrogen metabolism have not been systematically explored. In this research, we analyzed transcriptional profiles of rice GOGAT genes using a genome-wide microarray database, and investigated the effects of suppression of glutamate synthase genes on carbon and nitrogen metabolism using GOGAT co-suppressed rice plants. Transcriptional profiles showed that rice GOGAT genes were expressed differently in various tissues and organs, which suggested that they have different roles in vivo. Compared with the wild-type, tiller number, total shoot dry weight, and yield of GOGAT co-suppressed plants were significantly decreased. Physiological and biochemical studies showed that the contents of nitrate, several kinds of free amino acids, chlorophyll, sugars, sugar phosphates, and pyridine nucleotides were significantly decreased in leaves of GOGAT co-suppressed plants, but the contents of free ammonium, 2-oxoglutarate, and isocitrate in leaves were increased. We conclude that GOGATs play essential roles in carbon and nitrogen metabolism, and that they are indispensable for efficient nitrogen assimilation in rice.