Patatin represents a group of homologous primary storage proteins (with molecular weights ranging from 40 kDa to 45 kDa) found in Solanum tuberosum L. This group comprises 40% of the total soluble proteins in potato tubers. Here, patatin (40 kDa) was extracted from potato fruit juice using ammonium sulfate precipitation (ASP) and exposed to high hydrostatic pressure (HHP) treatment (250, 350, 450, and 550 MPa). We investigated the effect of HHP treatment on the structure, composition, heat profile, and antioxidant potential, observing prominent changes in HHP-induced patatin secondary structure as compared with native patatin (NP). Additionally, significant (p < 0.05) increases in β-sheet content along with decreases in α-helix content were observed following HHP treatment. Thermal changes observed by differential scanning calorimetry (DSC) also showed a similar trend following HHP treatment; however, the enthalpy of patatin was also negatively affected by pressurization, and free sulfhydryl content and surface hydrophobicity significantly increased with pressurization up to 450 MPa, although both interactions progressively decreased at 550 MPa. The observed physicochemical changes suggested conformational modifications in patatin induced by HHP treatment. Moreover, our results indicated marked enhancement of antioxidant potential, as well as iron chelation activities, in HHP-treated patatin as compared with NP. These results suggested that HHP treatment offers an effective and green process for inducing structural modifications and improving patatin functionality.