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The histology and, accordingly, biomechanics of the human corneal stroma are highly heterogeneous. There is extensive knowledge on the existence of regional differences in the collagen fibril bundles' packing arrangement and lamellar orientation across and throughout the thickness of the human corneal stroma.1–5 The anterior stroma consists of short bundles of collagen fibrils that show dense intertwining and also insert vertically into Bowman's layer, thus contributing to maintain corneal shape. The deeper stroma contains collagen fibril bundles that are arranged in wide lamellae oriented predominantly along the superior/inferior and nasal/temporal meridians and with reduced connections between adjacent layers. Several laboratory techniques have been used to elucidate the depth-dependent mechanical properties of the human corneal stroma.6–17 All of these studies have shown that the elastic modulus within the stroma decreases from anterior to posterior, regardless of the mechanical testing used. It is therefore well accepted that the depth-dependent changes of stromal mechanical elasticity are linked to the depth-dependent change of stromal microstructure. Atomic force microscopy (AFM) enables localized mechanical sample testing and has been used to investigate the local elastic modulus of the human corneal stroma at different depths. Although the results from previous studies6–11 could not be directly compared, because of the use of AFM tips with variable geometry and dimension as well as different study protocols, the elastic modulus is shown to decrease 40% to 80% with depth from the anterior stroma to the posterior part of the stroma. The aim of this study was to provide information on the depth-dependent compressive elastic modulus of the human corneal stroma both at the tissue (stroma) and molecular (collagen) level by using AFM. In addition, we correlated the depth-dependent anisotropy of stromal elasticity with images of the human corneal stroma acquired by second harmonic generation (SHG) microscopy.