Pebrine disease, caused by Nosema bombycis (Nb) infection in silkworms, is a severe and long-standing disease that threatens sericulture. As parasitic pathogens, a complex relationship exists between microsporidia and their hosts at the mitochondrial level. Previous studies have found that the translocator protein (TSPO) is involved in various biological functions, such as membrane potential regulation, mitochondrial autophagy, immune responses, calcium ion channel regulation, and cell apoptosis. In the present study, we found that TSPO expression in silkworms (BmTSPO) was upregulated following Nb infection, leading to an increase in cytoplasmic calcium, adenosine triphosphate, and reactive oxygen species levels. Knockdown and overexpression of BmTSPO resulted in the promotion and inhibition of Nb proliferation, respectively. We also demonstrated that the overexpression of BmTSPO promotes host cell apoptosis and significantly increases the expression of genes involved in the immune deficiency and Janus kinase-signal transducer and the activator of the transcription pathways. These findings suggest that BmTSPO activates the innate immune signalling pathway in silkworms to regulate Nb proliferation. Targeting TSPO represents a promising approach for the development of new treatments for microsporidian infections.