A high-quality inoculum of Gluconacetobacter xylinus is important to produce bacterial cellulose (BC), a versatile biomaterial. This work aims to develop a method of preparing an inoculum of this bacterium with high cell density and without mutants.Inocula of G. xylinus ACCC 10220 without and with cellulase or carboxymethyl cellulose (CMC) were prepared in shaken culture. BC pellets and BC-negative mutants were present in the inoculum without additives but absent in the inoculum with additives. Based on BC weights statically produced in fresh BC-producing media initiated by different seed culture, the 24-h-shaken inoculum with 1·50% (w/v) CMC was the best because of high biomass and absence of mutants. The BC weights in fresh media inoculated by the 96-h-static inoculum and 24-h-shaken CMC inoculum at 7% (v/v) were 0·70 and 1·05 g l(-1) , respectively, implying significant difference (P < 0·01) in BC weights. However, structure properties of the two BC samples, including the crystallinity index, mass fraction of cellulose Iα , degree of polymerization (DP) and micromorphology were slightly different.The 24-h-shaken CMC inoculum was the most suitable for a starter culture of BC.A novel method of preparing G. xylinus inoculum in shaken culture was developed, featuring high biomass, absence of mutants and no BC entanglements. Cellulase or CMC added into the medium completely suppressed mutation of G. xylinus, and CMC facilitated to form colloidal BC with the low DP in shaken culture, indicating less BC stress to cells. These findings suggested the mutation could be induced by BC stress, and not by shear stress commonly accepted.