Human pDC recognize Influenza virus, HIV-1 and small molecules from the imidazoquinoline family through TLR7 and HSV and CpGA through TLR9, resulting in production of type I and III IFN and pro-inflammatory cytokines. Imidazoquinolines have been used for anti-viral therapy and as immune adjuvants and are often considered to be prototypical TLR7 agonists. However, there have been reports of these molecules inhibiting virus-induced cytokine production. We investigated the mechanisms by which the imidazoquinoline, 3M003, induces type I and III IFN production in pDCs while inhibiting virus-stimulated cytokine production. 3M003 induced Type I and III IFN and TNF-ɑ much more rapidly than Flu or HSV, but at much lower levels and for shorter duration; actin polymerization and endosomal acidification were required for this activation. The TLR7 inhibitor IRS661 effectively inhibited Flu and 3M003-induced IFN production. 3M003 inhibited Flu, HSV, HIV and CpGA induced IFN production in pDC when used together with these TLR7/9 agonists. This inhibitory effect of 3M003 was observed when it was added any time in the first four hours of virus stimulation. Similar to Flu and HSV, 3M003 enhanced IRF7 serine 477/479 phosphorylation but, surprisingly, did not translocate IRF7 to the nucleus as shown by imaging flow cytometry. Instead, 3M003 and the related molecule, R848, strongly and rapidly (within 30 minutes) induced IRF5 nuclear translocation in pDC, while HSV induced strong IRF7 and to a similar extent, IRF5 translocation, but later. Co-culture of pDC with 3M003 and virus yielded the high IRF5/low IRF7 translocation phenotype. HSV-1- but not 3M003-induced IFN was inhibited by the PI3K inhibitor, LY294-002. Taken together, these results indicate a novel signaling pathway in pDCs wherein 3M003 or 3M003 plus virus preferentially activates IRF-5 in a PI3K-independent manner, while HSV activates both IRF-5 and -7, as well as PI3K, resulting in the different levels and kinetics of IFN production.