The Atomic Force Microscope tip was used to progressively abrade the surface of non-cutted starch granules embedded in the endosperm protein matrix in grain sections from wheat near-isogenic lines differing in the puroindoline b gene and thus hardness. In the hard near-isogenic wheat lines, starch granules exhibited two distinct profiles corresponding either to abrasion in the surrounding protein layer or the starch granule. An additional profile, only identified in soft lines, revealed a marked stop in the abrasion at the protein-starch transition similar to a lipid interface playing a lubricant role. It was related to the presence of both wild-type puroindolines, already suggested to act at the starch-protein interface through their association with polar lipids.This study revealed, for the first time, in situ differences in the nano-mechanical properties at the starch-protein interface in the endosperm of wheat grains depending on the puroindoline allelic status.