The effect of the sulfatide incorporation on the nanomechanical properties of pure dipalmitoylphosphatidylcholine (DPPC) vesicles was studied using atomic force microscope surface. The forces, measured between an AFM tip and the vesicle, presented that the breakthrough of the tip into the vesicles occurred two times. Each breakthrough represented each penetration of the tip into each bilayer. Force data prior to the first breakthrough were fitted well with the Hertzian model to estimate Young's modulus and bending modulus of the vesicles. It was found that the incorporation led to decrease by around 90% in Young's modulus and bending modulus of the vesicles. The decrease appears to be attributed to the disruption of DPPC headgroup packing, which is caused by the larger hydration shell around the sulfatide headgroup.