The chemokine RANTES (regulated upon activation, normal T-cells expressed and secreted) plays an essential role in inflammation and immune response. Infection with wild-type foot-and-mouth disease virus (FMDV) in PK-15 cells strongly inhibits the expression of RANTES compared to infection with a genetically engineered mutant lacking the leader protein (L(pro)) coding region. This suggests that L(pro) is involved in RANTES regulation. However, the underlying molecular mechanism remains unclear. In this study, we show that transfection of PK-15 cells with a plasmid expressing the L(pro) of FMDV, in the absence of other FMDV proteins, inhibited dsRNA-induced RANTES transcription and promoter activity. Promoter mutagenesis experiments revealed that the interferon-stimulated response element (ISRE) was important for the ability of L(pro) to inhibit dsRNA-induced RANTES promoter activity. Furthermore, over-expression of L(pro) also inhibited IRF-3/7-mediated RANTES activation. Screening L(pro) mutants indicated that catalytic activity and a SAP (for SAF-A/B, Acinus, and PIAS) domain of L(pro) were required to suppress dsRNA-induced RANTES transcription.