The effects of immunostimulatory RNAs (isRNAs) on the expression of immuno-suppressive factors are largely unknown. Indoleamine 2,3-dioxygenase (IDO) is a key negative regulator of immune responses and it has been implicated in hampering immunity against tumours. Here we show that the activation of Toll-like receptors (TLR)-7/8 with isRNAs or R848, a specific ligand for TLR7/8, can induce IDO expression in human monocytes, but not in monocyte-derived dendritic cells (moDC). In contrast to TLR7/8 agnosists, treatment of the same moDC with interferon-gamma-induced IDO expression. Treatment of monocytes with 2'-O-methyl uridine-modified isRNAs alone does not induce IDO, but totally abrogated the effects of unmodified isRNAs. Like isRNAs, synthetic viral RNAs and cytomegalovirus (CMV) induced IDO in monocytes, whereas TLR2 ligand lipopeptide Pam3Cys exhibited no effect. Furthermore, IDO positive monocytes suppressed autologous T-cell activation. Collectively, these data indicate for first time that the potency of TLR7/8 signalling pathways to induce IDO expression in monocytes is silenced when the cells are programmed to differentiate into dendritic cells. The immunosuppressive properties of IDO might confer an advantage to CMV-infected monocytes to escape T-cell responses. The findings that 2'-O-methyl modified RNAs can block isRNA-induced IDO expression would facilitate the design of new TLR inhibitors.