Endospores of the Gram-positive bacterium, Bacillus subtilis, have been used successfully for delivery of antigens where the immunogen is expressed on the spore surface. In this work the spore has been engineered to deliver antigens to the cytoplasm of macrophages by expressing listeriolysin O (LLO) or a derivative, LLOL461T, that is stable at neutral pH, from the B. subtilis vegetative cell. Following phagocytosis spores were shown to germinate in the phagosome enabling secretion of LLO/LLOL461T and entry of the bacterium into the cytosol. We have shown that in the cytosol B. subtilis proliferates before eventually being destroyed. Immunisation of mice with spores that co-expressed LLO with Protective Antigen (PA) of Bacillus anthracis generated an increase in IgG2a against PA, toxin-neutralising activity coupled with specific IFN-γ and IL-12 (and reduced IL-4) responses of splenocytes, both indicative of an enhanced Th1 response. Enhanced Th1 responses via LLO co-expression of antigen by B. subtilis spores may be a useful strategy to improve vaccine performance.