The innate immune response is the first line defense against viral infections. Novel genes involved in this system are continuing to emerge. SLC15A3, a proton-coupled histidine and di-tripeptide transporter that was previously found in lysosomes, has been reported to inhibit chikungunya viral replication in host cells. In this study, we found that SLC15A3 was significantly induced by DNA virus herpes simplex virus-1(HSV-1) in monocytes from human peripheral blood mononuclear cells. Aside from monocytes, it can also be induced by HSV-1 in 293T, HeLa cells, and HaCaT cells. Overexpression of SLC15A3 in 293T cells inhibits HSV-1 replication and enhances type I and type III interferon (IFN) responses, while silencing SLC15A3 leads to enhanced HSV-1 replication with reduced IFN production. Moreover, we found that SLC15A3 interacted with MAVS and STING and potentiated MAVS- and STING-mediated IFN production. These results demonstrate that SLC15A3 participates in anti-HSV-1 innate immune responses by regulating MAVS- and STING-mediated signaling pathways.