Poly(N-isopropyl-acrylamide) (PNIPAM) is a paradigm thermally sensitive polymer, which has a lower critical solution temperature (LCST) of 32 °C in water. Herein by AFM-based single molecule force spectroscopy (SMFS), we measured the single chain elasticity of PNIPAM across the LCST in water. Below LCST, the force curves obtained at different temperatures have no remarkable difference; while above LCST, an unexpected temperature dependent elasticity is observed, mainly in the middle force regime. We found that 35 °C is a turning point of the variation: from 31 to 35 °C, the middle parts of the force curves drop gradually, whereas from 35 to 40 °C, the middle parts rise gradually. A possible mechanism for the unexpected temperature dependent mechanics is proposed. The single chain contraction against external force upon heating from 35 to 40 °C may cast new light on the design of molecular devices that convert thermal energy to mechanical work.