We have performed a systematic backbone N-methylation of a 13-residue SIM peptide and assessed the effects on SUMO binding using a combination of competition fluorescence anisotropy and relaxation-based NMR experiments. At seven positions in the sequence, binding was abrogated or adversely affected, whereas at six positions binding was unaffected or improved. In instances where binding was diminished, this could be readily ascribed to the methylated peptide being unable to adopt a stable bound conformation. In instances where methylation led to improved binding potency, this could be ascribed to faster binding to SUMO. Interestingly, this occurred for both hot-spot and flanking residues from the SIM peptide.