The Interleukin-1/Toll-like receptor signaling pathway is a crucial signaling pathway within the innate immune system and the use of mass spectrometric techniques became valuable to investigate signal transduction pathways. To date only a few reports exist that focus on the mass spectrometric identification of novel signaling intermediates within the TLR signal transduction pathway. Here we used this approach systematically to identify new interaction partners of the TLR signaling pathway and subsequently characterized them functionally. We identified 14-3-3 theta as a new member of the TLR signaling complex. With genetic complementation assays, we demonstrate that 14-3-3 negatively regulates TLR2-dependent NF-κB activity and amplifies the TLR4-dependent activation of the transcription factor. While 14-3-3 has no effect on TLR-induced apoptosis in innate immune cells, it controls the release of the inflammatory, IRF3-dependent cytokines like RANTES and IP-10 after stimulation with LPS. Most strikingly, 14-3-3 controls the production of proinflammatory cytokines like IL-6, IL-8, and TNFα in a different manner. Our results identify 14-3-3 theta as a new and important regulatory protein in the TLR signaling suppressing the MyD88-dependent pathway.