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• Effect of heat-moisture treatment (HMT) on starch in adlay seeds were revealed. • HMT could alter the hierarchical structures of starch in adlay seeds. • HMT reduced the digested ratio and rate of starch in adlay seeds. • The new ordered structure formed during HMT weakened starch digestibility. • HMT effects on starch in adlay seeds are intensified by treatment moisture content. In the present study, starches isolated from heat-moisture treated (HMT) adlay seeds were characterized with hierarchical structures and digestibility to understand the relationships in structural and digestible behaviors of starches in a HMT starch-based food system. The results indicated that HMT not only caused the disorganizations of hierarchical structures, but also promoted the molecular rearrangements and reassembly of starch chains to form a new ordered molecular aggregation architecture (i.e. , highly ordered molecular structure, amylose-lipids complexes and thicker crystalline lamella). And the reinforced molecular interactions between starch molecules during HMT occurred to form more densely ordered structure, thus reducing the digestibility and digestion rate coefficients (k) of adlay starch. Particularly, HMT applied directly in adlay seeds with 30% moisture content displayed the highest resistant starch level (20.6%) and lowest k (2.74 × 10−3 min−1). Thus, the results are useful to realize the desired regulation of starch digestibility in adlay products following HMT. [ABSTRACT FROM AUTHOR]