We developed a simple preparation method for poly(N-isopropylacrylamide) (poly(NIPAM))-bonded silica using a water-organic two-phase system to directly copolymerize N-isopropylacrylamide with 3-(methacryloxy)propyl moieties on the silica surfaces. The stationary phase showed a phase transition with the change in temperature or in methanol content, resulting in notable changes in selectivity which cannot be expected with alkyl-type stationary phases. The packing material showed an increase in retention and column efficiency with the increase in temperature between 30 and 40 °C or in methanol content between 10 and 20%. The temperature-responsive properties were reduced with the increase in methanol content of the mobile phase and lost at 40% or higher. Significant changes in retention selectivity by the addition of methanol were observed for benzene derivatives below the transition temperature of poly(NIPAM). The preferential retention of hydrogen bond acceptors was observed at low temperatures in the mobile phase of low methanol content, while dominant contribution of the hydrophobic interaction to the retention of solutes was observed at the higher temperature in aqueous mobile phase or at the higher methanol concentration. The temperature increase brought about similar changes in retention behavior of the poly(NIPAM)-bonded silica packing materials to the addition of methanol to the mobile phase.