The regulation of the Il12b gene, encoding the shared p40 subcomponent for IL-12 and IL-23, is critical for innate immune responses and subsequent T cell polarization. This gene is robustly induced upon Toll-like receptor (TLR) stimulation, wherein an enhancer located 10 kb upstream of the transcription start site is required for promoter activity; however, the underlying mechanisms that regulate this enhancer in cooperation with the promoter has remained elusive. We show here that the IL12b enhancer contains functional ISREs for recognition by interferon regulatory factors (IRFs), and provide evidence that TLR-activated IRF5 mediates cooperativity of the enhancer with the promoter which also contains ISREs. By contrast, IRF3 activated by cytosolic RIG-I-like receptor (RLR) signaling binds to these ISREs and causes gene suppression. Consistently, IRF5 binding is accompanied with chromatin remodeling of both regulatory regions and the formation of a productive transcriptional complex containing other transcription factors, whereas these events are inhibited by IRF3 binding. We show that the ISREs embedded in the enhancer are indeed critical for its activation by IRF5. We also adduce evidence that the 5' sequences of the enhancer and promoter ISREs, all of which deviate from consensus ISREs, critically affect the function of IRF3. The dual commitment of these IRFs in the regulation of the Il12b enhancer and promoter is unique and may have implications for understanding the evolution of this gene. (C) 2012 Elsevier Inc. All rights reserved.