The structure of poly(N-isopropylacrylamide) (PNIPAM) in solution is still an unresolved topic. Here, the PNIPAM structure in water was investigated using a bottom-up approach, involving the monomer, dimer, and trimer, and a combination of infrared (IR) spectroscopies as well as molecular dynamics simulations. The experiments show that the monomer and oligomers exhibit a broad and asymmetric amide I band with two underlying transitions, while PNIPAM presents the same major transitions and a minor one. Analysis of the 2D IR spectra and theoretical modeling of the amide I band indicates that the two transitions of the monomer do not have the same molecular origin as the oligomers and the polymer. In the monomer, the two bands originate from the ultrafast rotation of its ethyl group, which leads to different solvation structures for the various rotational conformers. In the case of the oligomers, the asymmetry and splitting of the amide I band is caused by the vibrational coupling among adjacent amide side chains. Moreover, it is deduced from the simulations that the oligomers have three distinct backbone conformations for neighboring amides. In particular, two of the backbone conformations have a closed and compact structure, while in the third, the backbone is open and elongated. The bottom-up approach allowed us to infer that such backbone conformations exist in PNIPAM as well. Consequently, the two major amide I transitions of the polymer are also assigned to split amide I transitions resulting from the vibrationally coupled nearest-neighboring amides. In contrast, the additional minor transition observed in PNIPAM is assigned to unsolvated amide units of the polymer. The proposed molecular model successfully describes that PNIPAM amide I band changes with temperature in terms of its molecular structure. This new model strongly suggests that PNIPAM does not have a completely random backbone structure, but has distinct backbone conformers between neighboring amides.