Montmorillonite (MMT) nanosheets with large sizes is significant for controllable preparation of MMT functional nanocomposite materials. Compared with MMT, two-dimensional MMT nanosheets have a larger specific surface area, higher aspect ratio and richer hydroxyl groups at the edges. Therefore, MMT nanosheets are widely applied in the fields like environmental remediation, energy storage, flame retardant and membrane materials. The methods for preparing two-dimensional MMT nanosheets include mechanical milling, shearing, intercalation and ultrasonic exfoliation. However, the size of MMT nanosheets can be seriously damaged because of the higher external energy input of these exfoliation methods, thus affecting the applications. It is thus necessary to propose a new method for preparing MMT nanosheets with large sizes. In this paper, a method of MMT exfoliation was investigated based on its nature of osmotic hydration through weakening the interlayer binding energy of MMT. The exfoliation mechanism of MMT based on osmotic hydration was revealed via the conductivity analysis and molecular simulation. In addition, the regulation mechanism of exfoliation was also explored through the regulation of the water consumption and the concentration of MMT suspension during the osmotic hydration.