Glycerol would stimulate the production of poly(gamma-glutamic acid) (gamma-PGA) and decrease its molecular weight in Bacillus subtilis NX-2. When 20 g/l glycerol was added in the medium, the yield of gamma-PGA increased from 26.7 +/- 1.0 to 31.7 +/- 1.3 g/l, and molecular weight of gamma-PGA decreased from 2.43 +/- 0.07 x 10(6) to 1.86 +/- 0.06 x 10(6) Da. In addition, it was found that the decrease of gamma-PGA chain length by glycerol would lead to the decrease of broth viscosity during the fermentation and enhanced the uptake of substrates, which could not only improve cell growth but also stimulate gamma-PGA production. Moreover, it was also found that glycerol could effectively regulate molecular weight between 2.43 +/- 0.07 x 10(6) and 1.42 +/- 0.05 x 10(6) Da with the concentration ranging from 0 to 60 g/l. This was the first time to discover such contribution of glycerol on gamma-PGA production in Bacillus genus. And the effects of glycerol on molecular weight of gamma-PGA would be developed to be an approach for the regulation of microbial gamma-PGA chain length, which is of practical importance for future commercial development of this polymer.