Display omitted •Succinylation of OPI could decrease zeta potential and increase molecular weight.•Acetylation and succinylation could significantly affected H0 of OPI.•Succinylation led to remarkable increase in the molecular weight of OPI.•Acylation could result in the transformation of β-sheet to α-helix and random coil.•OPI had less compact tertiary conformation by acylation, especially succinylation. The effects of acetylation and succinylation on physicochemical properties and structural characteristics of oat protein isolate (OPI) were investigated. The degree of N-acylation rapidly increased prior to O-acylation, due to higher reactivity of ε-amino groups than hydroxyl groups. The acylation was able to decrease zeta potential of OPI at neutral pH, and succinylated OPI had lower zeta potential than acetylated OPI. The surface hydrophobicity (H0) of OPI was changed significantly by acylation treatment, which varied with the type and level of applied anhydrides. The succinylation led to a remarkable increase in the molecular weight of OPI determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The secondary structure and tertiary conformation of proteins in the OPI was analyzed by Fourier transform infrared (FTIR) and intrinsic fluorescence spectroscopy. The acylation could result in the transformation of β-sheet to α-helix and random coil, and less compact tertiary conformation, especially succinylation.