Leprosy is caused mainly by Mycobacterium leprae, a rod-shaped bacillus that is an obligate intracellular (only grows inside of certain human and animal cells) bacterium. M. leprae is termed an "acid fast" bacterium because of its chemical characteristics. When special stains are used for microscopic analysis, it stains red on a blue background due to mycolic acid content in its cell walls. The Ziehl-Neelsen stain is an example of the special staining techniques used to view the acid-fast organisms under the microscope.

Currently, the organisms cannot be cultured on artificial media. The bacteria take an extremely long time to reproduce inside of cells (about 12-14 days as compared to minutes to hours for most bacteria). The bacteria grow best at 80.9 F-86 F, so cooler areas of the body tend to develop the infection. The bacteria grow very well in the body's macrophages and Schwann cells (cells that cover and protect nerve axons). M. leprae is genetically related to M. tuberculosis (the type of bacteria that cause tuberculosis) and other mycobacteria that infect humans. As with malaria, patients with leprosy produce anti-endothelial antibodies (antibodies against the lining tissues of blood vessels), but the role of these antibodies in these diseases is still under investigation.

In 2009, investigators discovered a new Mycobacterium species, M. lepromatosis, which causes diffuse disease (lepromatous leprosy). This new species (determined by genetic analysis) was found in patients located in Mexico and the Caribbean islands.

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