Glial scar is a dense scar tissue that forms at the site of injury in the central nervous system. Although it is known that microglia, which are immune cells in the brain, are important in the formation of glial scar, the molecular mechanism is still unclear. It has been reported that the expression of TRPV4, a multistimulus receptor, is commonly upregulated in various injured areas. In this study, we focused on TRPV4 in microglia to understand the mechanism of glial scar formation. First, to investigate the effect of microglial TRPV4 on glial scar formation, we used a glial scar model using cultured hippocampal sections. Microglia-specific knockdown of TRPV4 reduced GFAP density in astrocytes, the main component of glial scar. Thus, microglial TRPV4 promotes glial scar formation. The abundance of TRPV4 in microglial lysosome of wild-type mice suggested that TRPV4 may regulate lysosomal function. To test this possibility, we conducted pharmacological studies using primary cultures of microglia, and found that treatment with GSK1016790A, a TRPV4 activator, increased cell surface LAMP1, suggesting that TRPV4 activation enhances lysosomal exocytosis in microglia. These results suggest that microglia promote glial scar formation by lysosomal exocytosis via TRPV4 activation.