Atomic force microscopy (AFM) can not only obtain topographical information of samples, but also measure their mechanical properties at an atomic resolution. This leads to a better understanding of pathophysiological mechanisms of human diseases because the alterations of topographical information and biomechanical properties of cells and molecules can be correlated to diseases. In recent years, AFM has been widely used in cell biological studies and study of diseases like diabetes mellitus, showing great potential in the early diagnosis and treatment guidance of the diseases. This review mainly summarized the application of AFM in the pathogenetic studies of diabetes and its related complications.