The purpose of this in vitro study was to evaluate the efficacy of a new resin emulsion (Pain-Free Desensitizer) treatment for dentine hypersensitivity, for its ability to decrease dentine permeability. Crown segments were prepared from extracted, unerupted human 3rd molars by horizontal sectioning to remove occlusal enamel and the roots. The specimens were allocated in one of two groups: In group 1, the dentine surface was acid-etched to simulate the patent tubules of hypersensitive dentine. In group 2, the mineralized dentine surface was polished free of smear layer using a hydroxyapatite paste and ultrasonication. The hydraulic conductance of each specimen was then measured to obtain a pretreatment of control value. After a single treatment with resin desensitizer, the permeability was remeasured at 5 min, 1 day, 1 week and 1 month. Between measurements, the specimens were stored in buffer solution to simulate the solubilizing effects of saliva. Parallel specimens were followed by SEM examination. The results showed that a single treatment with resin desensitizer produced large, immediate, reductions in dentine permeability in both acid-etched and mineralized surfaces. In the acid-etched (group 1) specimens, the permeability returned to control values within 7 days, while the permeability of the group 2 specimens remained low even after 30 days of soaking. This simple treatment for occluding dentinal tubules may provide sufficient temporary reduction in dentine permeability to permit the development of natural desensitization.