HIV-1-infected macrophages are long-lived and act as human immunodeficiency virus 1 (HIV-1) virus reservoirs. Lipopolysaccharide (LPS) has been demonstrated to suppress HIV-1 replication in macrophages, but the mechanism is not clear. Previous research suggested that downregulation of CD4 and CCR5 as well as blockage of the interaction of HIV-1 with cells are major causes of inhibition of HIV-1 replication in macrophages by LPS. In order to study whether LPS blocks the post-entry event of HIV-1 replication, we developed a macrophage HIV-1 infection model by using VSV-G pseudotyped HIV-1-luciferase virus to infect THP-1 differentiated macrophage-like cells. We found that LPS can suppress HIV-1 replication at post-entry steps. Further study suggested that HIV-1 reverse transcription was blocked by LPS, but addition of exogenous deoxyribonucleosides led to only partial recovery of HIV-1 replication. However, the inhibition of pro-inflammatory pathway completely rescued HIV-1 replication. Thus, our study shows that LPS can suppress the events of HIV-1 replication post-entry, including reverse transcription, and this restriction is mediated by more than one mechanism.