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With in situ atomic force microscopy (AFM) imaging, the structure of nanoparticles assembled at the water-oil interface is clearly resolved with the nanometer in 3D. The increase in the surfactant concentration in the oil phase leads to the increase in the contact angle of the nanoparticles at the water-oil interface. Unlike the prediction by equilibrium theory, a 2D monolayer to a 3D multilayer transition of nanoparticles is observed at high surfactant concentrations, which is attributed to the co-existence of completed wetted and partially wetted nanoparticles at the water-oil interface. The contact angle change and structural transition of nanoparticles induced by the increase of surfactant concentration is further confirmed macroscopically by the phase-inversion of Pickering emulsions and anomalous compliance of liquid interface. Our study sheds light on the importance of both thermodynamics and kinetics on the …