Japanese encephalitis virus (JEV) is a leading cause of viral encephalitis. However, the mechanisms of JEV penetration of the blood-brain-barrier (BBB) remain poorly understood. Mast cells (MCs) are granulated innate immune sentinels located perivascularly, including at the BBB. Here we show that JEV activates MCs, leading to the release of granule-associated proteases in vivo. MC-deficient mice display reduced BBB permeability during JEV infection compared to congenic wild-type (WT) mice, indicating that enhanced vascular leakage in the brain during JEV infection is MC-dependent. Moreover, MCs promoted increased JEV infection in the central nervous system (CNS), enhanced neurological deficits, and reduced survival in vivo. Mechanistically, chymase, a MC-specific protease, enhances JEV-induced breakdown of the BBB and cleavage of tight-junction proteins. Chymase inhibition reversed BBB leakage, reduced brain infection and neurological deficits during JEV infection, and prolonged survival, suggesting chymase is a novel therapeutic target to prevent JEV encephalitis.