The demulsification mechanism of asphaltene-stabilized water-in-toluene emulsions by an ethylene-oxidepropylene oxide (EOPO) based polymeric demulsifier was studied. Demulsification efficiency was determined by bottle tests and correlated to the physicochemical properties of asphaltene interfacial films after demulsifier addition. From bottle tests and droplet coalescence experiments, the demulsifier showed an optimal performance at 2.3 ppm (mass basis) in toluene. At high concentrations, the demulsification performance deteriorated due to the intrinsic stabilizing capacity of the demulsifier, which was attributed to steric repulsion between water droplets. Addition of demulsifier was shown to soften the asphaltene film (i.e., reduce the viscoelastic moduli of asphaltene films) under both shear and compressional interfacial deformations. Study of the macrostructures and the chemical composition of asphaltene film at the toluenewater interface after demulsifier addition demonstrated gradual penetration of the demulsifier into the asphaltene film. Demulsifier penetration in the asphaltene film changed the asphaltene interfacial mobility and morphology, as probed with Brewster angle and atomic force microscopy.