AbstractCollagen fibrils serve as the molecular scaffolding for connective tissues throughout the human body and are the most abundant protein building block in the extracellular matrix (ECM). Glycosaminoglycans (GAGs) are an important class of polysaccharides in the ECM that mediate collagen fibril development, cell adhesion and motility, wound healing, inter alia. Depletion or mis-regulation of GAGs has been shown to be intimately related to diseases such as cancer, hyperglycemia, and glaucoma. However, the molecular-level coordination between collagen fibrils and GAGs to regulate ECM mechanobiology is unclear. Here, high resolution atomic force microscopy experiments are used to assess perturbations to nanoscale topography and mechanical properties of single collagen fibrils upon GAG depletion in aqueous solution. Fast force maps show that GAG depletion lowers indentation modulus by about half compared to native fibrils while retaining a key structural motif (D-banding). These results shed new light on the functional role of GAGs and may aid in strategies to treat diseases that impair ECM mechanobiology.