We show that the protein encoded by the glutamine synthetase translational inhibitor (gstI) gene reduces the NH₄⁺ assimilation capacity of Rhizobium leguminosarum. In this organism, gstI expression is regulated by the ntr system, including the P^II protein, as a function of the nitrogen (N) status of the cells. The GstI protein, when expressed from an inducible promoter, inhibits glutamine synthetase II (glnII) expression under all N conditions tested. The induction of gstI affects the growth of a glutamine synthetase I (glnA^-) strain and a single amino acid substitution (W48D) results in the complete loss of GstI function. During symbiosis, gstI is expressed in young differentiating symbiosomes (SBs) but not in differentiated N₂-fixing SBs. In young SBs, the P_II protein modulates the transcription of NtrC-regulated genes such as gstI and glnII. The evidence presented herein strengthens the idea that the endocytosis of bacteria inside the cytoplasm of the host cells is a key step in the regulation of NH₄⁺ metabolism.