The mechanism and molecular structure of the slowly digestible waxy maize starch prepared by octenyl succinic anhydride (OSA) esterification and heat-moisture treatment were investigated. The in vitro Englyst test showed a proportion of 28.3% slowly digestible starch (SDS) when waxy maize starch was esterified with 3% OSA (starch weight based, and it is named OSA-starch), and a highest SDS content of 42.8% was obtained after OSA-starch (10% moisture) was further heated at 120 degrees C for 4 h (named HOSA-starch). The in vivo glycemic response of HOSA-starch, which showed a delayed appearance of blood glucose peak and a significant reduction (32.2%) of the peak glucose concentration, further confirmed its slow digestion property. Amylopectin debranching analysis revealed HOSA-starch had the highest resistance to debranching enzymes of isoamylase and pullulanase, and a simultaneous decrease of K-m and V-m (enzyme kinetics) was also shown when HOSA-starch was digested by either alpha-amylase or amyloglucosidase, indicating that the slow digestion of HOSA-starch resulted from an uncompetitive inhibition of enzyme activity during digestion. Size exclusion chromatography analysis of HOSA-starch showed fragmented amylopectin molecules with more nonreducing ends that are favorable for RS conversion to SDS by the action of amyloglucosidase in the Englyst test. Further solubility analysis indicates that the water-insolubility of HOSA-starch is caused by OSA-mediated cross-linking of amylopectin and the-hydrophobic interaction between OSA-modified starch molecules. The water-insolubility of HOSA-starch would decrease its enzyme accessibility, and the digestion products with attached OSA molecules might also directly act as the uncompetitive inhibitor to reduce the enzyme activity leading to a slow digestion of HOSA-starch.