Significance A key enzyme in inorganic nitrogen assimilation, glutamine synthetase, is the target of multiple regulatory mechanisms. Here we describe the nitrogen stress-induced RNA 4 (NsiR4), a small regulatory RNA that reduces the expression of inactivating factor 7 (IF7), an inhibitory factor of glutamine synthetase in cyanobacteria. The expression of NsiR4 is under positive control through the nitrogen control transcription factor (NtcA). NtcA also induces the transcription of the glutamine synthetase gene and represses the gene encoding IF7. Therefore, NsiR4 is a new player in the NtcA-mediated regulation of nitrogen assimilation, which is important for adaptations to rapid changes in available nitrogen sources and concentrations.