Abstract Background Nitrogen is essential for microbial growth and its importance is demonstrated by the complex regulatory systems used to control the transport, assimilation and metabolism of nitrogen. Recent studies are beginning to shed light on how mycobacteria respond to nitrogen limitation and several regulators (e.g., GlnR, P II ) have been characterized at a molecular level. However, despite this progress, our knowledge of the transcriptional response of mycobacteria to nitrogen limitation and its regulation is confined to batch culture. Methods To gain further insight into the response of mycobacteria to nitrogen limitation, we developed a nitrogen-limited chemostat. We compared the transcriptional response of nitrogen-limited cells to carbon-limited cells using RNA-seq analysis in a continuous culture model at a constant growth rate. Conclusions Our findings revealed significant changes in the expression of 357 genes (208 upregulated, 149 downregulated; >2-fold change, false discovery rate