Detailed measurements of cell material properties are required for understanding how cells respond to their mechanical environment. Atomic force microscopy (AFM) is an increasingly popular measurement technique that uniquely combines subcellular mechanical testing with high-resolution imaging. However, the standard method of analyzing AFM indentation data is based on a simplified “Hertz” theory that requires unrealistic assumptions about cell indentation experiments. The objective of this study was to utilize an alternative “pointwise modulus” approach, that relaxes several of these assumptions, to examine subcellular mechanics of cultured human aortic endothelial cells (HAECs). Data from indentations in 2‐to5‐μm square regions of cytoplasm reveal at least two mechanically distinct populations of cellular material. Indentations colocalized with prominent linear structures in AFM images exhibited depth-dependent variation of the apparent pointwise elastic modulus that was not observed at adjacent locations devoid of such structures. The average pointwise modulus at an arbitrary indentation depth of was and (, ) for these two material populations, respectively. The linear structures in AFM images were identified by fluorescence microscopy as bundles of f-actin, or stress fibers. After treatment with cytochalasin B, HAECs behaved like a homogeneous linear elastic material with an apparent modulus of . These findings reveal complex mechanical behavior specifically associated with actin stress fibers that is not accurately described using the standard Hertz analysis, and may impact how HAECs interact with their mechanical environment.