Lysosomal stress due to the accumulation of nucleic acids (NAs) activates endosomal TLRs in macrophages. Here, we show that lysosomal RNA stress, caused by the lack of RNase T2, induces macrophage accumulation in multiple organs such as the spleen and liver through TLR13 activation by microbiota-derived ribosomal RNAs. TLR13 triggered emergency myelopoiesis, increasing the number of myeloid progenitors in the bone marrow and spleen. Splenic macrophages continued to proliferate and mature into macrophages expressing the anti-inflammatory cytokine IL-10. In the liver, TLR13 activated monocytes/macrophages to proliferate and mature into monocyte-derived KCs (moKCs), in which, the liver X receptor (LXR) was activated. In accumulated moKCs, tissue clearance genes such as MerTK, AXL, and apoptosis inhibitor of macrophage (AIM) were highly expressed, while TLR-dependent production of proinflammatory cytokines was impaired. Consequently, Rnaset2-/- mice were resistant to acute liver injuries elicited by acetaminophen (APAP) and LPS with D-galactosamine. These findings suggest that TLR13 activated by lysosomal RNA stress promotes the replenishment of tissue-protective Kupffer cells.