Epstein-Barr virus (EBV) infection contributes to tumorigenesis of various human malignancies including nasopharyngeal carcinoma (NPC). EBV triggers innate immune and inflammatory responses partly through Toll-like receptor (TLR) signaling. Lactoferrin (LF), with its anti-inflammatory properties, is an important component of the innate immune system. We previously reported that LF protects human B lymphocytes from EBV infection by its ability to bind to the EBV receptor CD21, but whether LF can suppress EBV-induced inflammation is unclear. Here, we report that LF reduced synthesis of IL-8 and monocyte chemoattractant protein-1 (MCP-1) induced by EBV in macrophages via its suppression of NF-κB activity. LF interacted with TLR2 and interfered with EBV-triggered TLR2-NF-κB activation. LF inhibited the ability of TLR9 to recognize dsDNA by binding to its co-receptor CD14, which blocked the interaction between CD14 and TLR9. EBV-induced inflammation was thus aggravated in the presence of CD14. In addition, LF expression levels were significantly downregulated in NPC specimens, and correlated inversely with IL-8 and MCP-1 expression. These findings suggest that LF may suppress the EBV-induced inflammatory response through interfering with the activation of TLR2 and TLR9.