Herpes simplex virus type-1 (HSV-1) is wide-spread dsDNA virus that establishes life-long latency and causes diverse severity of symptoms. In this study, HEK 293T cells with low Toll-like receptor (TLR) and Stimulator of interferon genes protein (STING) expression was infected with HSV-1 and subjected to quantitative proteomic analysis. By using a subcellular fractionation strategy and high-performance mass spectrometry, a total of 5,982 host proteins were quantified, of which 484 proteins were differentially regulated. Bioinformatics analysis indicated that multiple signaling pathways were involved in HSV-1 infection.