Polyrotaxanes, a family of rod-shaped nanomaterials comprised of noncovalent polymer/macrocycle assemblies, are being used in a growing number of materials and biomedical applications. Their physiochemical properties can vary widely as a function of composition, potentially leading to different in vivo performance outcomes. We sought to characterize the pharmacokinetic profiles; toxicities, and protein corona compositions of 2-hydroxypropyl-beta-cyclodextrin polyrotaxanes as a function of variations in macrocycle threading efficiency, molecular weight, and triblock copolymer core structure.-We show that polyrotaxane fate in vivo is governed by the structure and,dynamics of their rodlike morphologies, such that highly threaded polyrotaxanes are long circulating and deposit in the liver, whereas lung deposition and rapid clearance is observed for species bearing lower 2-hydroxypropyl-beta-cyclodextrin threading percentages. Architecture differences also promote recruitment of different serum protein classes and proportions; however, physiochemical differences have little or no influence on their toxicity. These findings provide important structural insights for guiding the development of polyrotaxanes as scaffolds for biomedical applications.