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Zinc signals are induced in cells of the immune system following the activation of several receptors. One of these receptors is the toll-like receptor (TLR)4. Subsequently to TLR4 activation, an increase in free Zn2+ is required for the MyD88-dependent expression of proinflammatory cytokines. The aim of this study was to compare the effect of Zn2+ on the MyD88 and TRIF-dependent signaling pathways in the murine macrophage cell line RAW 264.7. A reduction of the intracellular available Zn2+ with the chelator N,N,N´,N´-tetrakis-(2-pyridyl-methyl)ethylenediamine (TPEN) reduces the mainly MyD88-dependent transcription of the cytokines interleukin (IL)-1beta, IL-6 and IL-10. Concomitantly, there is an increase in TRIF-dependent transcription of interferon (IFN)-beta and the IFN-beta-dependent inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) release. This effect is independent of the TLR4-induced zinc signal and is also observed after stimulation of TLR3, the only TLR that does not induce a zinc signal and signals solely via TRIF. Following the stimulation of TLR4, basal Zn2+, instead of the zinc signal, is a negative regulator of the TRIF-dependent signaling pathway by inhibiting interferon regulatory factor (IRF)3-dependent transcription. Furthermore, the expression of the transcription factor signal transducer and activator of transcription (STAT)1 in the IFN-beta-induced JAK-STAT signaling pathway is decreased by TPEN indicating a possible feedback mechanism on elevated iNOS expression. The cytokine IFN-gamma also induces the expression of iNOS. IFN-gamma does not induce a zinc signal and the NO release is independent of incubation with TPEN. Hence, the impact of the basal Zn2+-concentration is specific for TLR signaling pathways. Here, zinc signals regulate the MyD88-dependent pathway and basal Zn2+ levels alter the TRIF-dependent pathway. Altogether, Zn2+ influences the balance of the two main TLR signaling pathways that are important for an adequate activation of innate immunity.