The microsporidian Enterocytozoon bieneusi is an obligate intracellular pathogen that causes enteric disease (microsporidiosis) in humans and has been recorded in a wide range of animal species worldwide. The transmission of E. bieneusi is direct and likely occurs from person to person and from animal to person via the ingestion of spores in water, food, or the environment. The identification of E. bieneusi is usually accomplished by molecular means, typically using the sequence of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA. Currently, ~820 distinct genotypes of E. bieneusi have been recorded in at least 210 species of vertebrates (mammals, birds, reptiles, and amphibians) or invertebrates (insects and mussels) in more than 50 countries. In this chapter, we provide a perspective on (1) clinical aspects of human microsporidiosis; (2) the genome and DNA markers for E. bieneusi as well as molecular methods for the specific and genotypic identification of E. bieneusi; (3) epidemiological aspects of E. bieneusi of animals and humans, with an emphasis on the genotypes proposed to be zoonotic, human-specific, and animal-specific; and (4) future research directions to underpin expanded molecular studies to better understand E. bieneusi and microsporidiosis.