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The midgut of bees plays a significant role in digestion and absorption and is a gateway for pathogens. As a protection for the organism, the midgut lumen is lined by a peritrophic matrix rich in chitin fibrils, associated with proteins that form multiple resistant layers with gelatinous consistency, with several functions, including the mechanical barrier against infection by pathogens. The obligate intracellular microsporid Nosema ceranae is an important pathogen that decreases honey production, increases pesticide susceptibility, and may cause mortality in the honey bee Apis mellifera. This study evaluated whether the peritrophic matrix has a protective function against infection by N. ceranae in A. mellifera adult workers. The peritrophic matrix integrity was changed by feeding the bees with calcofluor white (CFW), followed by an artificial infection with N. ceranae spores. Results shown that 0.5% CFW affect the integrity of the peritrophic matrix with areas of interruption of this envelope. This disorganization of the peritrophic matrix reduces the life cycle period of N. ceranae from eight to four days, after inoculation, and increases the concentration of new spores released in the midgut lumen. This is the first report on the importance of the peritrophic matrix as a protective barrier against N. ceranae infection in A. mellifera.