During neurotropic virus infection, microglia act as a source of chemokines, thereby regulating the recruitment of peripheral leukocytes and the multicellular immune response within the CNS. Herein, we present a comprehensive study on the chemokine production by microglia in response to double-stranded RNA (dsRNA), a conserved molecular pattern of virus infection. Transcriptional analyses of chemokine genes revealed that dsRNA strongly induces the expression of CXC chemokine ligand 10 (CXCL10) and CC chemokine ligand 5 (CCL5) in microglia. We also observed that the dsRNA stimulation triggered the activation of signaling pathways mediated by nuclear factor kappaB (NF-kappaB) and mitogen-activated protein kinases (MAPK), including extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38, and c-Jun N-terminal kinase (JNK). The microglial CXCL10 response to dsRNA was induced via NF-kappaB, p38, and JNK pathways, whereas the dsRNA-induced CCL5 production was dependent on JNK, but not on the other signal-transducing molecules tested. In addition, the acidic environment of intracellular vesicles was required for the activation of cellular signaling in response to dsRNA. Taken together, these results suggest that the recognition of dsRNA structure selectively induces the CXCL10 and CCL5 responses in microglia through vacuolar pH-dependent activation of NF-kappaB and MAPK signaling pathways.