The peritrophic matrix (PM) secreted by the midgut cells of insects is formed by the binding of PM proteins to chitin fibrils. The PM envelops the food bolus, serving as a barrier between the content of the midgut lumen and its epithelium, and plays a protective role for epithelial cells against mechanical damage, pathogens, toxins, and other harmful substances. However, few studies have investigated the characteristics and synthesis factors of the PM in the silkworm, Bombyx mori. Here, we examined the characteristics of the PM in the silkworms. The PM thickness of the silkworms increased gradually during growth, while there was no significant difference in thickness along the entire PM region. Permeability of the PM decreased gradually from the anterior to posterior PM. We also found that PM synthesis was affected by food ingestion and the gut microbiota. Our results are beneficial for future studies regarding the function of the PM in silkworms.