Pea starches, in both native (NPS) and retrograded-autoclaved forms (RAPS), were subjected to simulated gastrointestinal (GI) digestion in vitro, their multi-scale structural characteristics, morphological features, molecular distribution and thermal properties were characterized. A gradual increase in the short-/long-range crystallinity, melting enthalpy of gelatinization on increasing digestion time was observed for both the native and retrograded-autoclaved pea starch samples based on the X-ray diffraction, Fourier-transform infrared spectra, solid-state CNMR and differential scanning calorimetry measurements. It was especially noticed that the growth rate of crystallinity and double helices, as well as the decrease in M values were evidently greater for RAPS than for NPS. To investigate how different molecular fine structure of pea starch substrate affects the gut microbiota shifts and dynamic short-chain fatty acid profile, their resistant starch residues obtained from both native and retrograded-autoclaved pea starch after 8 h of simulated GI tract digestion was used as the fermentation substrate. The levels of acetate, propionate and butyrate gradually increased with the increasing fermentation time for NPS and RAPS. In comparison to the blank control (i.e., the group without the addition of carbohydrate), the fermented NPS and RAPS obviously resulted in an increased abundance of Firmicutes and Bacteroidetes, accompanied by a decrease in Proteobacteria, Actinobacteria and Verrucomicrobia. Both NPS and RAPS promoted different shifts in the microbial community at the genus level, with an increase in the abundance of Bacteroides, Megamonas and Bifidobacterium, as well as a reduction in the abundance of Fusobacterium, Faecalibacterium and Lachnoclostridium in comparison to the blank control samples.