Fever onset is correlated with viremia in dengue virus (DENV) patients. Heat shock factor 1 (HSF1), a heat stress response host transcription factor, plays a crucial role in regulating multiple cellular functions, as well as the onset of infectious diseases. This study evaluated the role of HSF1 in DENV replication as a means of regulating DENV infection in vitro and in vivo. DENV infection activated HSF1 in both Ca2+ and protein kinase A-dependent manners. Inhibiting HSF1 effectively reduced DENV replication, not only in THP-1 cells but also in primary human monocytes. Activated HSF1 contributed to DENV replication by upregulating autophagy-related protein (Atg) 7, as autophagy is crucial for virus replication. Heat stress also activated HSF1, which in turn facilitated DENV replication. Activated HSF1, the increased Atg7, and autophagic induction were founded in the DENV-infected brains and pharmacologically inhibiting HSF1 reduced autophagy, viral protein expression, neuropathy, and mortality. These results provide new insight into HSF1 as a novel host factor for DENV infection through its role in facilitating autophagy-regulated viral replication in the brains.