Poly(N-isopropylacrylamide) (PNIPAAm) grafted onto silica, which may be used for reverse phase chromatography (RPC), was simulated and synthesized for protein separation with temperature-triggered adsorption and desorption. Molecular dynamics simulation at an all-atom level was performed to illustrate the adsorption/desorption behavior of cytochrome c, the model protein, on PNIPAAm-grafted-silica, a temperature responsive adsorbent. At a temperature above the lower critical solution temperature (LCST), the PNIPAAm chains aggregate on the silica surface, forming a hydrophobic surface that is favorable for the hydrophobic adsorption of cytochrome c, which has a high exposure of hydrophobic patches. At temperatures below the LCST, the PNIPAAm chains stretch, forming hydrophilic surface due to hydrogen bonding between PNIPAAm and surrounding water. Desorption of cytochrome c on the PNIPAAm-grafted-silica surface occurs as a result of competition with water, which forms hydrogen bonds with the protein. The conformational transitions of both cytochrome c and PNIPAAm are monitored, providing molecular insight into this temperature-responsive RPC technique. PNIPAAm-grafted-silica beads were synthesized and used for the adsorption and desorption of cytochrome c at approximately 313 K and 290 K, respectively. The experimental results validate the molecular dynamics simulation. In comparison to conventional RPC, using temperature as a driving force for RPC reduces the risk of protein denaturation caused by exposure to chaotropic solvents. Moreover, it simplifies the separation process by avoiding the buffer exchange operations between the steps.