During macrophage activation, expression of IL-1R-associated kinase (IRAK)-M is induced to suppress TLR-mediated responses and is a hallmark of endotoxin tolerance. Endotoxin tolerance requires tight regulation of genes occurring at the transcriptional and epigenetic levels. To identify novel regulators of IRAK-M, we used RAW 264.7 macrophages and performed a targeted RNA interference screen of genes encoding chromatin-modifying enzymes, signaling molecules, and transcription factors involved in macrophage activation. Among these, the transcription factor CCAAT/enhancer binding protein (C/EBP)β, known to be involved in macrophage inactivation, was necessary for the induction of IRAK-M expression. Chromatin immunoprecipitation showed that C/EBPβ was recruited to the IRAK-M promoter following LPS stimulation and was indispensable for IRAK-M transcriptional activation. Among histone 3-modifying enzymes, our screen showed that knockdown of the histone 3 lysine 27 (H3K27) methyltransferase and part of the polycomb recessive complex 2, enhancer of Zeste 2, resulted in IRAK-M overexpression. In contrast, knockdown of the H3K27 demethylase ubiquitously transcribed tetratricopeptide repeat X chromosome suppressed the induction of IRAK-M in response to LPS stimulation. Accordingly, we demonstrated that H3K27 on the IRAK-M promoter is trimethylated in unstimulated cells and that this silencing epigenetic mark is removed upon LPS stimulation. Our data propose a mechanism for IRAK-M transcriptional regulation according to which, in the naive state, polycomb recessive complex 2 repressed the IRAK-M promoter, allowing low levels of expression; following LPS stimulation, the IRAK-M promoter is derepressed, and transcription is induced to allow its expression.