Hypothesis: Amphiphilic Janus nanosheets are plate-shaped, with one hydrophilic and one hydrophobic side; they are expected to assemble at oil-water interfaces. The assembled Janus nanosheets layers at the oil-water interface will exhibit a unique mechanical response under the vertical pressure of a probe. Experiments: The interfacial behaviors of amphiphilic Janus nanosheets and the morphology of the assembled particle film at an oil-water interface were observed. The dynamic morphologies and force displacement curves of the oil-water interface covered with amphiphilic Janus nanosheets were investigated during the insertion of a cylindrical probe. Findings: Amphiphilic Janus nanosheets spontaneously aggregated at the oil-water interface. The morphology of the assembled particle film was controlled by the interfacial nanosheets concentration and can be divided into three regimes: unsaturated, monolayer, and collapsed. The wettability of the probe and the density of nanosheets at the interface played critical roles in the deformation and mechanical response of the oil-water interface under vertical pressure. The presence of amphiphilic Janus nanosheets reduced the stiffness and enhanced the flexibility and deformability of the oil-water interface. The oil water interface covered with amphiphilic Janus nanosheets could produce larger deformation under a smaller vertical stress. This work not only improves the understanding of the interfacial properties of amphiphilic Janus nanosheets but also provides a method for characterizing nanoparticle layers at oil water interfaces. (C) 2020 Elsevier Inc. All rights reserved.