Mucus is a complex biological material that lubricates and protects the human lungs, gastrointestinal (GI) tract, vagina, eyes, and other moist mucosal surfaces. Mucus serves as a physical barrier against foreign particles, including toxins, pathogens, and environmental ultrafine particles, while allowing rapid passage of selected gases, ions, nutrients, and many proteins. Its selective barrier properties are precisely regulated at the biochemical level across vastly different length scales. At the macroscale, mucus behaves as a non-Newtonian gel, distinguished from classical solids and liquids by its response to shear rate and shear stress, while, at the nanoscale, it behaves as a low viscosity fluid. Advances in the rheological characterization of mucus from the macroscopic to nanoscopic levels have contributed critical understanding to mucus physiology, disease pathology, and the development of drug delivery systems designed for use at mucosal surfaces. This article reviews the biochemistry that governs mucus rheology, the macro- and microrheology of human and laboratory animal mucus, rheological techniques applied to mucus, and the importance of an improved understanding of the physical properties of mucus to advancing the field of drug and gene delivery.