ABSTRACT Background Major peanut allergens are stable and resistant to denaturation under standard cooking conditions, contributing to allergenicity and low rates of developing natural tolerance in allergic individuals. We evaluated the effects of heat and pressure autoclaving on peanut proteins, IgE binding, and oral tolerability. Methods Raw, roasted, and autoclaved peanut protein extracts were evaluated by Bradford assay, ELISA, and mass spectrometry‐proteomics to compare relative amounts of protein, specific IgE binding, and allergen fragmentation. To assess changes in clinical reactivity, we performed skin prick testing (SPT) in 41 subjects using standard and autoclaved peanut extracts. We also performed double‐blind oral food challenges (OFC) in 10 peanut‐allergic subjects with standard and autoclaved peanuts. Results Autoclaving at 130°C, 2.4 atm, for 30 min significantly degraded allergens Ara h 1 and 2, and completely degraded Ara h 8. Mass spectrometry‐proteomics analysis of size‐filtered extracts (< 10 kDa) showed greater numbers and diversity of peptides from peanut proteins and allergens in autoclaved extracts. Autoclaving fragmented proteins into shorter peptides, against which sera from highly allergic patients exhibited a 74% reduction in IgE binding compared to raw peanuts. SPT demonstrated significant decreases in wheal diameters using autoclaved peanut extract (median [IQR] = 5 mm [2, 9]) compared to commercial extract (10 mm [6, 15]; p < 0.001). All OFC subjects tolerated the maximum cumulative autoclaved peanut dose (444 mg) versus standard peanut (median: 9 mg, range: [1, 44]). Conclusions Autoclaving peanuts induces important chemical changes including fragmentation, leading to decreased peanut allergenicity and consequently increased tolerability. This has the potential for novel immunotherapeutic approaches with more favorable side effect profiles.