The effect of acylation with various short-chain fatty acids on starch fine structure, digestion and gut microbiota fermentation property was investigated. Nuclear magnetic resonance spectra of acylated starch convinced the existence of acyl protons. Consistently, Fourier transform infrared spectra showed a carbonyl C[dbnd]O vibration at 1730 cm−1 due to the introduced acyl groups. X-ray diffraction (XRD) and small angle X-ray scattering (SAXS) studies revealed that the acylation destroyed the internal structure for providing a chance of rearrangement of starch molecules with the formation of a different fractal structures. More interestingly, compared with native starch, there were a higher Δρu and a lower Δρ for acylated starches, suggesting more ordered aggregation structures was formed inside the starch granules. Kinetics of in-vitro hydrolytic enzymatic model and Pearson correlation coefficients further confirmed the association between multi-scale structural order and digestion characters. Acyl groups introduced by acetylation, propionylation and butyrylation onto the starch molecules could be effectively released by the intestinal flora during the fermentation, specifically increasing their corresponding SCFAs production, respectively. The greater generation of the specific SCFA from the acylated starches following the microbiota fermentation may highlight their importance in the application in food and pharmaceutical industry.