OPN, a cytokine produced, among others, by DCs, is involved in inflammation and defense against pathogens. Here, we report that the activation of the MyD88 pathway by TLR2, TLR5, and TLR7/8 agonists or IL-1β induces high levels of OPN in human DCs. Conversely, LPS and Poly I:C, two TLR3 and TLR4 agonists that engage the TRIF pathway, were ineffective. TLR2 agonists were the strongest OPN inducers, and OPN production was highly stimulated by TLR2-triggering bacteria (Staphylococcus aureus) but not by TLR4-triggering Escherichia coli. Costimulation experiments revealed that TLR3 and TLR4 agonists, beyond being inactive by themselves, sharply limited TLR2-dependent OPN production by activating a TRIF-dependent inhibition of the MyD88-dependent OPN production. MyD88 silencing impaired TLR2-dependent OPN induction, whereas TRIF pathway blockage by chloroquine, dynasore, or TRIF knockdown prevented the TLR3/4 agonist-mediated inhibition, which was independent from the endogenous production of type I IFN, IL-29, IL-10, or TGF-β. LPS and Poly I:C inhibitory activity was associated with the release of a >10-kDa protein factor(s). We also demonstrated that the higher OPN levels produced by S. aureus-treated DCs compared with E. coli-treated DCs were responsible for a markedly increased production of IL-17 by CD4⁺ T cells. These results highlight the biological relevance of the differential OPN induction by TLR2 and TLR4 agonists and emphasize the importance of TLR cross-talk in OPN induction. This implies that OPN regulation by TLR signaling is critical in shaping inflammatory responses and may modulate IL-17 production in response to pathogens.