Mucus is a complex biological material that lubricates and protects various moist mucosal surfaces in the human body, including the lungs, gastrointestinal tract, vagina, and eyes. It acts as a physical barrier against pathogens, toxins, and environmental particles while allowing the passage of selected substances. Mucus exhibits non-Newtonian behavior at the macroscale, behaving like a gel, and low viscosity at the nanoscale. Understanding mucus rheology is crucial for drug delivery systems targeting mucosal surfaces. This review discusses the biochemistry governing mucus rheology, macro- and microrheology of human and animal mucus, and the importance of understanding mucus physical properties for drug and gene delivery. Mucus is composed of mucins, DNA, lipids, ions, proteins, and cells, with mucins being the primary structural component. Mucin content and glycosylation influence mucus viscoelasticity, which is regulated by biochemical factors. DNA and lipids also contribute to mucus rheology, while salts and proteins affect its viscoelastic properties. Microrheology studies reveal that mucus microviscosity is similar to water at length scales up to 55 nm, but increases at larger scales. Understanding mucus microrheology is essential for designing therapeutic nanoparticle systems for mucosal delivery. Macrorheology of mucus is characterized by a non-Newtonian, shear-thinning behavior, with viscosity varying with shear rate and frequency. Mucus from different mucosal surfaces, such as respiratory, gastrointestinal, and cervicovaginal, exhibits distinct rheological properties. Macrorheology is important for understanding lung function, mucociliary transport, and disease pathology. Animal mucus models are valuable for studying mucus properties when human mucus is not available. Mucus from dogs, pigs, and rats has been characterized, showing similar viscoelastic behavior to human mucus. Understanding mucus rheology has implications for disease treatment, including the use of mucolytics to reduce mucus viscosity in conditions like cystic fibrosis.Mucus is a complex biological material that lubricates and protects various moist mucosal surfaces in the human body, including the lungs, gastrointestinal tract, vagina, and eyes. It acts as a physical barrier against pathogens, toxins, and environmental particles while allowing the passage of selected substances. Mucus exhibits non-Newtonian behavior at the macroscale, behaving like a gel, and low viscosity at the nanoscale. Understanding mucus rheology is crucial for drug delivery systems targeting mucosal surfaces. This review discusses the biochemistry governing mucus rheology, macro- and microrheology of human and animal mucus, and the importance of understanding mucus physical properties for drug and gene delivery. Mucus is composed of mucins, DNA, lipids, ions, proteins, and cells, with mucins being the primary structural component. Mucin content and glycosylation influence mucus viscoelasticity, which is regulated by biochemical factors. DNA and lipids also contribute to mucus rheology, while salts and proteins affect its viscoelastic properties. Microrheology studies reveal that mucus microviscosity is similar to water at length scales up to 55 nm, but increases at larger scales. Understanding mucus microrheology is essential for designing therapeutic nanoparticle systems for mucosal delivery. Macrorheology of mucus is characterized by a non-Newtonian, shear-thinning behavior, with viscosity varying with shear rate and frequency. Mucus from different mucosal surfaces, such as respiratory, gastrointestinal, and cervicovaginal, exhibits distinct rheological properties. Macrorheology is important for understanding lung function, mucociliary transport, and disease pathology. Animal mucus models are valuable for studying mucus properties when human mucus is not available. Mucus from dogs, pigs, and rats has been characterized, showing similar viscoelastic behavior to human mucus. Understanding mucus rheology has implications for disease treatment, including the use of mucolytics to reduce mucus viscosity in conditions like cystic fibrosis.