Monocytes/macrophages are known to represent a potential reservoir of human immunodeficiency virus type 1 (HIV‐1), which ensures continuous replication of the virus in patients on highly active antiretroviral therapy (HAART). Infected macrophages are a highly productive source of HIV‐1 during infections with common opportunistic pathogens. Previous studies report that toll like receptors (TLR)s play a role in HIV‐1 replication in macrophages. Here, we investigate the three main pathways activated through TLR4 and the interactions with the HIV‐1 long terminal repeat (LTR), using human embryonic kidney (HEK) 293 cells expressing TLR4 and transfected with a luciferase reporter under the control of the HIV‐1 LTR. Here, we demonstrate, that TLR4‐mediated activation of HIV‐LTR is largely governed by the nuclear factor‐κB pathway. Neither of the mitogen‐activated protein kinases ERK1/2, JNK, or p38 nor the transcription factor interferon regulatory factor 3 were involved in the direct transactivation of HIV‐LTR through stimulation of TLR4.