High pressure processing of ovotransferrin was carried out to study the structural and physiochemical changes of ovotransferrin under various pressure levels. At pH 8 and pressures higher than 200 MPa, a decrease in total sulfhydryl groups and an increase in surface hydrophobicity were observed along with a partial aggregation. A gradual shift of denaturation peak towards higher temperature was noticed up to 500 MPa, leading to a total loss of the enthalpy of denaturation at pressures of 600 and 700 MPa, where a significant decrease in intrinsic fluorescence was also observed. At pH 3, the ovotransferrin adopted a molten globule state, associated with a significant increase in surface hydrophobicity and reactive sulfhydryl content; structurally, no clear denaturation peaks in differential scanning calorimetry (DSC) were detected at any level of pressure treatment whereas a noticeable decrease in intrinsic fluorescence was evidenced up to 600 MPa and then increased at 700 MPa pressure treatment. Fourier transform infrared spectroscopy (FT-IR) revealed that the conformational structure were changed from helices, sheets, turns, and aggregated strand to mostly intermolecular β-sheets or aggregated strands at pH 8 at 200 MPa but switched back to original structure at higher pressures.