Natural Killer T (NKT) cells are lipid-reactive, CD1d-restricted T lymphocytes important in infection, cancer, and autoimmunity. In addition to foreign antigens, NKT cells react with endogenous self lipids. However, in the face of stimulating self antigen, it remains unclear how overstimulation of NKT cells is avoided. We hypothesized that constantly degraded endogenous antigen only accumulates upon inhibition of alpha-galactosidase A (alpha-Gal-A) in lysosomes. Here, we show that alpha-Gal-A deficiency caused vigorous activation of NKT cells. Moreover, microbes induced inhibition of alpha-Gal-A activity in antigen-presenting cells. This temporary enzyme block depended on Toll-like receptor (TLR) signaling and ultimately triggered lysosomal lipid accumulation. Thus, we present TLR-dependent negative regulation of alpha-Gal-A as a mechanistic link between pathogen recognition and self lipid antigen induction for NKT cells.