In present work, we elucidate the GlcNAc metabolic machinery in strain and demonstrated the role of nagA in GlcNAc metabolism by cloning a DNA fragment encoding a nagA and subsequently generating a nagA-deficient mutant by homologous recombination. The resulting knockout strain was examined for growth, cytoplasmic UDPGlcNAc pool, and overall cellulose productivity with glucose and/ or GlcNAc as a carbon source. The successful deletion of this gene and the subsequent analysis provides a clearer picture of the related metabolic pathways of this potentially important biosynthetic pathway. To determine whether nagA is essential for growth of the bacterium, both DnagA and wild type G. xylinus were grown for five days on HS agar plate supplemented with either glucose, GlcNAc, or glucosamine. On glucose and glucosamine supplemented plates, both mutant and wild type grew well while on GlcNAc supplemented plates, mutant cells did not grow. To confirm these data, the growth of both wild type and mutant cells was monitored in liquid HS media supplemented with glucose or GlcNAc. In glucose-supplemented media, both mutant and wild type cells exhibited a typical sigmoidal growth pattern whereas in the presence of GlcNAc, growth of mutant cells was completely inhibited while wild type cells grew slowly as expected due to the fact that GlcNAc is not a preferred carbon source for G. xylinus. Additionally, in presence of glucosamine, growth of both wild type and DnagA mutant was similar to the growth of wild type in GlcNAc fed conditions. These findings together with agar plate growth studies demonstrated that nagA disruption prevents G. xylinus from metabolizing GlcNAc as an alternative carbon source for growth. Similar findings were also observed in Gluconacetobacter intermedius where disruption of nagA decreased the growth rate in the exponential growth phase. Nevertheless, a steady sate growth curve of mutant cells with GlcNAc feed also revealed that nagA disruption does not cause any lethal impact on the bacterial cells and as a result it only impairs the growth. To conform the mutant cells were sub-cultured in the presence of glucose and found that bacterium regains its normal growth.