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The effect of urea on the phase transition of PNIPAM was studied using differential scanning calorimetry (DSC). For a certain urea concentration, the enthalpy change of phase transition of poly(N-isopropylacrylamide) (PNIPAM) aqueous solution increases with the number of DSC cycles, presumably due to the displacement of water molecules bound to the amide groups of PNIPAM by urea molecules at the temperature higher than the lower critical solution temperature (LCST) of PNIPAM and causes the decrease in the absolute value of the exothermic heat related to the dehydration of hydrophilic groups and interactions of hydrophilic residues to around 0. Moreover, the enthalpy change decreases with the urea concentration during the heating process of the first DSC cycle, indicating the replacement of water molecules around the apolar isopropyl groups by urea molecules at the temperature lower than LCST, and the endothermic heat caused by the dehydration of apolar groups decreases. Furthermore, the urea molecules which replace the water molecules at high temperature can be replaced again by water molecules at the temperature lower than LCST, but this process needs several days to complete.