Summary Two different Toll‐like receptors (TLRs) have been shown to play a role in host responses to Leishmania infection. TLR‐2 is involved in parasite survival in macrophages upon activation by lipophosphoglycan (LPG), a virulence factor expressed by Leishmania. In contrast, activation of TLR‐9 has been shown to promote a host‐protective response. However, whether there is a relationship between the interaction of LPG and TLR‐2, on one hand, with the effect of TLR‐9, on the other hand, remains unknown. In this study, we report that in‐vitro infection of macrophages with a L. major parasite with high expression levels of LPG results in decreased TLR‐9 expression compared to infection with a L. major parasite with lower expression levels of LPG. Addition of anti‐LPG as well as anti‐TLR‐2 antibodies prevents this reduction of TLR‐9 expression. Also, the addition of purified LPG to macrophages results in a decrease of TLR‐9 expression, which is shown to be mediated by transforming growth factor (TGF)‐β and interleukin (IL)‐10. Finally, in‐vitro treatment of macrophages with anti‐LPG and/or anti‐TLR‐2 antibodies before infection reduces the number of amastigotes in macrophages and co‐treatment of mice with anti‐TLR‐2 antibodies and cytosine–phosphate–guanosine (CpG) reduces footpad swelling and parasite load in the draining lymph nodes, accompanied by an interferon (IFN)‐γ‐predominant T cell response. Thus, for the first time, we show how interactions between LPG and TLR‐2 reduce anti‐leishmanial responses via cytokine‐mediated decrease of TLR‐9 expression.