TNF-alpha is a pivotal cytokine whose overproduction can be lethal. Previously, we identified a transcription factor, LPS-induced TNF-alpha factor (LITAF), that regulates TNF-alpha transcription. We now report the discovery and characterization of a regulatory cofactor that we call signal transducer and activator of transcription (STAT) 6(B) because of its considerable homology to STAT6 [here referred to as STAT6(A)]. The STAT6(B) gene expression was found to be activated by LPS. Furthermore, we show that cotransfection of STAT6(B) and LITAF induces an interaction between the two proteins, consequently forming a complex that subsequently translocates into the nucleus and up-regulates the transcription of cytokines. The effect of the complex on a panel of cytokines was tested. In addition, the specific role of LITAF in this complex was established with experiments, including RNA interference technology. Overall, these findings describe roles for LITAF, STAT6(B), and the LITAF-STAT6(B) complex in the regulation of inflammatory cytokines in response to LPS stimulation in mammalian cells.