The purpose of this study focuses on modifying β-LG (β-lactoglobulin) in buttermilk via supercritical CO₂ treatment, to reduce its antigenicity. In dairy industry, supercritical fluid extraction (SFE) is applied primarily in fat extraction. Yet it is possible to utilize SFE as a reactor in foods. Milk is an essential energy source for infants. However, formula is still needed in some instances and there are some children allergic to dairy based products. In milk proteins, β-LG is believed to be a prevalent allergen in cow’s milk. Buttermilk powder was used because of its abundant milk fat globule membrane and phospholipids content. It was treated in SFE system with food grade CO₂ at 100, 150, 200, 250, 350, 400 bars, with 50 and 75°C in each condition. All analyses were completed in 10% buttermilk suspension (wt/vol). Buttermilk proteins were examined in SDS-PAGE, Western-blot, and ELISA. Orbitrap Fusion HPLC-MS/MS and periodic staining were used to check any post-translational modification. Purified β-LG was used to evaluate the cytotoxicity, viability, and inflammatory response in Caco-2 cell line by the mean of LDH, MTT, and IL-8, respectively. The SDS-PAGE quantification showed that the signal intensity of β-LG bands was reduced in a maximum of 45.8% after being processed under 250 bar/75°C, 30 min. Lighter and diffused signals were also found in Western-blot. ELISA tests proved that our treatment can significantly change the β-LG antigenicity in buttermilk (P < 0.05). Around 146-Da molecular weight increment was detected on treated β-LG by Orbitrap Fusion. Sugar moieties in glycoproteins were revealed by periodic staining. Treated β-LG showed 95% viability and 27% lower inflammation without any cytotoxicity in Caco-2 cells, compared with untreated β-LG. These results showed that β-LG reduction was completed by glycosylation, which has been reported as a possible pathway to reduce the allergenicity in foods. The denaturation of β-LG in supercritical fluid processing is a promising way to solve milk allergy, which is still a problem requiring more attention and further research.