Normal maize starch and high amylose maize starch (HAS) either in native or thermally treated forms were used to investigate the effect of starch structure on the production of metabolites and gut microbiota profile using an anaerobic in vitro system. The changes in starch structure during fermentation were investigated using scanning electron microscopy (SEM), high-performance liquid chromatography (HPLC) and Fourier transform infra-red spectroscopy (FTIR). The native normal starch showed a porous structure during fermentation, indicating it was quickly metabolized by gut bacteria, whereas the HAS showed a smooth structure, suggesting it was utilized gradually. HPLC chromatography showed that amylose fraction with low molecular weight (MW) had a higher resistance to be fermented by gut bacteria than other starch molecular fractions. Thermal treatment enhanced starch fermentation kinetics, especially for amylopectin and high MW amylose fractions. FTIR analysis suggests that the structure of the normal starch, either in native or thermally treated, was less organized compared to HAS, and this structural character led to the normal starch to be utilized more quickly by gut bacteria with a faster increase in the IR ratio 1047/1022 cm(-1) (P < 0.01) during fermentation. The measurement of metabolic activity indicates that the normal starch with a less organized structure was utilized faster and generated more acetate and lactate during fermentation; HAS with a highly organized structure was more likely to produce butyrate, corresponding the significant increase (P < 0.001) in the populations of butyrate-producing strains (Faecalibacterium prausnitzii and Eubacterium hallii) in the cultures. This study reveals that fermentation kinetics of starch substrate is one of important characteristics for manipulating gut microbiota fermentation behaviours.