The purpose of this study is to elucidate the effect of molecular architecture of grafted polymers on the temperature-responsive hydrophilic/hydrophobic alterations of poly(N-isopropylacrylamide) (PIPAAm)-modified surfaces as well as the interaction with hydrophobic steroids. In particular, we focus on the effect of freely mobile PIPAAm chains introduced onto PIPAAm looped chain grafted surfaces. For this purpose, we prepared following two types of PIPAAm-modified surfaces; 1) looped chain grafted surfaces using poly (IPAAm-co-N-acryloxysuccinimide), 2) PIPAAm terminally-grafted surfaces using residual active eater groups on the looped chain grafted surfaces. Temperature-responsive surface property changes were evaluated by means of dynamic contact angle measurements. Different temperature-responsive surface wettability changes were observed for these surfaces. Surface wettability changed drastically at 305 K for the surface with freely mobile PIPAAm chains, the temperature corresponds to the LCST of PIPAAm in water. However, the PIPAAm looped chain grafted surface showed a lower transition temperature than the surface immobilized freely mobile PIPAAm chains. The difference in graft architecture also influenced the retention behavior of steroids with different hydrophobicities. At higher temperature, separation of steroids on both surfaces was accomplished in aqueous media. The surface with freely mobile PIPAAm chains showed a longer retention time than the PIPAAm looped chain grafted surface. This is due to a stronger hydrophobic interaction between the surface with freely mobile PIPAAm chains and steroid molecules. Consequently, PIPAAm graft architecture on the surface strongly influenced both wettability changes and interaction with steroids in response to temperature changes. These results suggest that we could regulate temperature-responsive hydrophobic interactions with solutes by designing PIPAAm molecular architecture on the surface of chromatography matrix.