This study investigates the evolution of the distributions of whole molecular size and of chain length of granular wheat starches (37 ~ 93% amylose content), subjected to in vitro fermentation with a porcine faecal inoculum or digestion with pancreatic enzymes. The results showed that the molecular structures of high-amylose starch (HAS) unfermented residues largely remained unchanged during the fermentation process, while wild-type starch (37% amylose content) showed a preferential degradation of the amylopectin fraction. In contrast, under simulated digestion conditions, the undigested residues of HAS showed structural changes, including a decrease in amylose content, a shift of amylose peak position towards lower degrees of polymerisation, and an enzyme-resistant fraction. These changes of starch structure are likely to be dependent on the different starch-degrading enzyme activities present in pancreatic vs. microbial systems. Molecular changes in response to fermentation metabolism revealed by size-exclusion chromatography can help understand the microbial utilization of resistant starch.