This paper presents a method for single cell stiffness measurement based on a nano-needle and nanomanipulation. The nano-needle with a buffering beam was fabricated from an atomic force microscope cantilever by the focused ion beam etching technique. Wild type yeast cells (W303) were prepared and placed on the sample stage inside an environmental scanning electron microscope (ESEM) chamber. The nanomanipulator actuated the nano-needle to press against a single yeast cell. As a result, the deformation of the cell and nano-needle was observed by the ESEM system in real-time. Finally, the stiffness of the single cell was determined based on this deformation information. To reveal the relationship between the cell stiffness and the environmental humidity conditions, the cell stiffness was measured at three different humidity conditions, i.e. 40, 70 and 100%, respectively. The results show that the stiffness of a single cell is reduced with increasing humidity.