The recognition of single-stranded RNA by TLR7/8 leads to the production of NF-kappa B-mediated cytokines and type I IFNs. However, the role of TLR7/8 activation in monocytes and macrophages is still unclear. The aim of this study was to investigate the differences in the activation of TLR7/8 between these two cell types. Microarray analysis, qRT-PCR and flow cytometry were used to analyse TLR7/8 signalling pathways in monocytes and macrophages after stimulation with agonists. Our data indicated that TLR8 agonists activated the NF-kappa B- and IRF-mediated pathways in THP-1 cells, whereas TLR7 agonists did not. However, silent TLR8 and enhanced TLR7 expression could increase TLR7-induced NF-kappa B activation in monocytes. TLR7 and TLR8 agonists induced NF-kappa B activation but no ISG response in PMA-differentiated THP-1 cells. The mRNA levels of pro-inflammatory cytokine were elevated upon CL075 stimulation in macrophages compared to monocytes. Thus, TLR7 and TLR8 might modulate different immune responses in monocytes and macrophages.