Nucleotides participate in various physiological processes through their supramolecular interactions with biomolecules. Therefore, the molecular recognition of nucleotides became an important topic in supramolecular chemistry and exhibited many biomedical applications. Guanidinocalixarenes showed very strong binding affinities towards nucleotides, even reaching the nanomolar level. In this work, we systematically determined the binding constants between a typical guanidinocalixarene(guanidinium-modified calix[5]arene, GC5A) and various nucleotides and revealed the driving forces behind the molecular recognition using theoretical calculations. The electrostatic interactions and hydrogen bonding provided by the phosphate groups of the nucleotides dominated the binding between the nucleotides and GC5A. The lower rim alkyl chains and the skeleton of GC5A provide preorganized cavity and upper guanidinium groups. The difference in the type of nucleobase is also attributed to the different binding affinities. This work provides insight into the molecular recognition of nucleotides and facilitates the development of new supramolecular hosts for nucleotides and related biological applications.