cAMP Receptor Protein (CRP) is a global regulatory protein in Escherichia coli which plays an important role in carbon catabolite repression. In this investigation, isothermal titration calorimetry (ITC) was used to analyze the binding affinity between CRP and an inhibitory peptide known as SpfP. Post-hoc analysis reveals a relatively weak binding affinity in the micromolar range, providing new insights into the molecular mechanism by which SpfP regulates CRP activity.