InStreptomyces coelicolor,amtBtranscription is promptly regulated by the global nitrogen regulator GlnR. Although the GlnR bindingcis-element has been characterized inamtBpromoter, consisting of three GlnR boxes ofa3-b3,a1-b1, anda2-b2, its role in GlnR-mediated transcriptional regulation remains unclear. Here, we showed that GlnR had different binding affinity against each pair of GlnR binding sites inamtBpromoter (i.e.,a3-b3,a1-b1, anda2-b2sites), and GlnR was able to binda3-b3anda1-b1, respectively, but nota2-b2alone. Consistently,a2was not a typical GlnR binding site and further experiments showed thata2was non-essential for GlnR-mediated bindingin vitroand transcriptional regulationin vivo. To uncover the physiological role of the three GlnR boxes, we then mutated the wild-typeamtBpromoter to a typical GlnR-binding motif containing two GlnR boxes (a3-b3-a2-b2), and found although the transcription of the mutated promoter could still be activated by GlnR, its increasing rate was less than that of the wild-type. Based on these findings, one could conclude that the three GlnR boxes assisted GlnR in more promptly activatingamtBtranscription in response to nitrogen limitation, facilitating bacterial growth under nitrogen stresses.