Hydrogen bonding (HB) is vital for the phase transition and physical properties of stimuli responsive copolymers. However, the governing mechanism remains unclear. Herein, we investigated the HB dynamics in a representative thermo-responsive copolymer (PNIPAM-AM) aqueous solution. Three types of HB were identified: polymer-polymer chain (CO:H–N), polymer chain-water molecule (CO:H–O) and water-water molecules (H–O:H–O) by vibrational spectroscopy. Increasing polymer concentration, the H–O:H–O softened and CO:H–O stiffened, leading to the destruction of HB network in water-water clusters. Upon increasing solution temperature, CO:H–O softened while CO:H–N stiffened, resolving the dehydration and cross-linking of polymer chains. Confinement of water molecules in the “cages” (∼ tens to hundreds of micrometers) after sol-gel phase transition was visualized, disclosing the mechanism of water holding capacity of PNIPAM-AM. The correlation of molecule vibration and copolymer microscopic structure was established, contributing to the fundamental understanding of phase behavior and physical properties regulations in stimuli responsive copolymer aqueous solution.