Spherelike cellulose formation as a function of agitated culture rotational speeds and flask sizes for two different cellulose producing Acetobacter xylinum strains, JCM 9730 (ATCC 700178) and NCIMB (ATCC 23769), has been studied in this work. Results showed that the JCM 9730 strain could form spherelike cellulose particles in the agitated culture with a rotational speed above 100 rpm. The NCIMB strain, however, formed no spherelike cellulose particles under any culture condition examined. For the JCM 9730 strain, approximately 10 mm diameter spheres were produced at a rotational speed of 150 rpm in 100 mL of culture solution in a 150 mL Erlenmeyer flask, while 0.5-1 mm diameter particles were produced in 100 mL of agitated culture with a rotational speed of 200 rpm in a 250 mL Erlenmeyer flask. Data from the measurement of biomass concentration and bacterial cellulose concentration revealed that the JCM 9730 strain exhibited higher cellulose yield (up to 6.8 times) as compared to the NCIMB strain. Scanning electron microscopy analysis of lyophilized spherelike cellulose particles indicated that culture rotational speed had an impact on the internal structure of the spherelike particles. Smaller spherelike particles produced at 150 rpm were hollow and the cellulose shell exhibited a layered structure. Larger particles produced at 125 rpm were solid where the cellulose in the central region did not exhibit a layered structure, but the outer layer was similar in structure to the particles produced at 150 rpm.