The interaction between nucleotide molecules and lipid molecules plays important roles in cell activities, but the molecular mechanism is very elusive. In the present study, a small but noticeable interaction between the negatively charged phosphatidylethanolamine (PE) and Guanosine monophosphate (GMP) molecules was observed from the PE monolayer at the air/water interface. As shown by the sum frequency generation (SFG) spectra and Pi-A isotherm of the PE monolayer, the interaction between the PE and GMP molecules imposes very small changes to the PE molecules. However, the Brewster angle microscopy (BAM) technique revealed that the assembly conformations of PE molecules are significantly changed by the adsorption of GMP molecules. By comparing the SFG spectra of PE monolayers after the adsorption of GMP, guanosine and guanine, it is also shown that the hydrogen bonding effect plays an important role in the nucleotide-PE interactions. These results provide fundamental insight into the structure changes during the nucleotide-lipid interaction, which may shed light on the molecular mechanism of viral infection, DNA drug delivery, and cell membrane curvature control in the brain or neurons.