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As a kind of stimuli-responsive materials, the disulfide-containing material has received a tremendous amount of interest. A novel functional disulfide-containing waterbe polyurethane (DS-WPU) nanoemulsion was prepared via a one-step in situ phase inverse emulsification technique and its reducing-responsive property was investigated using dithiothreitol as the reductant. Results showed that the DS-WPU nanoemulsion had a uniform particle size in nanoscale and a good film-forming characteristic, and the DS-WPU latex film exhibited great responsiveness along with a self-assembled behavior in the reducing environment to form WPU emulsion again, including a long-to-short process of the polymer chains.