The objective of the paper is to examine in detail the relationship between the structure of starch-based nano-biocomposite materials (containing 0, 3 and 6 wt% of native sodium, and organo-modified montmorillonites with cationic starch) and their moisture barrier properties (permeability, water sorption and diffusivity). The structure was characterized by TEM, WAXD and DMA, whereas moisture transfer was evaluated thanks to permeability measurements and sorption kinetics analysis using a dynamic vapour sorption equipment. It was shown that the relatively high plasticizer content (23 wt% glycerol) induces a phase separation, with plasticizer rich and carbohydrate rich phases, resulting in the nanoclay being preferentially located in the carbohydrate rich domains. As a consequence, a preferential way for water transfer was more likely created in the very hydrophilic glycerol rich domains where the nanoclay platelets were almost totally absent. Thus, even if exfoliated morphology is achieved, the heterogeneous clay distribution and phase separation phenomena explain the lack of improvement and even the decline in the moisture barrier properties for these glycerol-plasticized starch nano-biocomposites.