The peanut de-oiling industry currently lacks efficient processing technologies for de-oiling at low or room temperatures. A novel method, micro-aqueous extraction (MAE), offers over 93 % de-oiling efficiency at room temperature and is also effective for other oilseeds like sesame, camellia, and rapeseed. Despite its effectiveness, the exact mechanism behind oleosomes destabilization at a critical hydration level or oil volume fraction (φ ∼ 0.75) is not fully understood. This study investigates how MAE affects peanut oleosome size, paste stability, and the interfacial properties of surfactant proteins. Results showed that micro-aqueous hydration and agitation caused small droplets (85.6 vol% < 10 μm) to coalesce into larger droplets (90.0 vol% > 30 μm) due to press-induced rupture of the liquid film. Simultaneously, agitation decreased water mobility and protein intrinsic fluorescence, while increasing paste viscosity, leading to protein aggregation. This aggregation further promoted oleosome coalescence. Additionally, hydration and agitation weakened the ability of membrane proteins to stabilize oleosomes by increasing interfacial tension and decreasing dilatational storage modulus. The insights into the peanut oleosome destabilization mechanism for MAE provide a foundation for scaling up the process, with the potential to replace current hot and cold pressing techniques.