As an extension study, FTIR and molecular simulation methods were combined in the present paper to analyze the H-bond interactions resulting from multiple donors and acceptors that have led to self-assembly based on segmented polyurethane with carboxyl (PUc) and poly(4-vinylpyridine) (P4VP) in our previous work. Of them, FTIR was used to analyze the H-bonding types and interactions as well as their changes before and after self-assembly; molecular mechanics (MM/COMPASS) was used to study the effect of possible conformations on the H-bonds involved and analyze the most probable H-bond patterns; quantum mechanics (QM/B3LYP) was used to help confirm the experimental FTIR band assignments and calculate the H-bond energy. It was found that two types of H-bonds exist, namely, COOH center dot center dot center dot P4VP (type I) and (OCO)NH center dot center dot center dot P4VP (type II), based on OH and NH as the strong donors in the interaction between PUc and P4VP. Strong evidence has been obtained for a type 11 H-bond, which is the specialty in PUc/P4VP assembly.. The type I and type 11 H-bonding energies are -11.293 and -7.150 kcal/mol, respectively. The forming probability of the type I H-bond accounts for 95.87%, while that of the type 11 H-bond is 4.13%, showing the primary driving force for the assembly based on PUc and P4VP is still the H-bond between COON and P4VP, yet the H-bonds based on NH and pyridyl in P4VP cannot be ignored.