The effect of high-frequency oscillatory type ball-mill treatment on the structure and antiobesity activity of konjac flour was investigated. The grain size of konjac flour changed from 657.3 microm (d(50)) to 23.7 microm (d(50)) after 4 h of treatment. The structural change of the konjac flour with different grain size was characterized by using X-ray diffraction (XRD) differential scanning calorimetry (DSC). The results indicated that the crystallinity decreased and the diffraction peak drifted not only by when the crystallization region was reduced but also when the crystalline structure was changed. With the decrease of the grain size and crystallinity, the konjac flour grain, especially the 4 h milled konjac flour, swelled more rapidly and led to the improvement of the antiobesity effect. Compared with the native konjac flour, the 4 h milled konjac flour could significantly decrease the body weight and total wet weight of fat of nutritional obese rats (P < 0.05) and also decreased the contents of triglyceride, glucose, and high-density lipoprotein in blood of nutritional obese rat significantly (P < 0.05), which meant the grain-size effect of konjac flour improved its antiobesity activity notably.