CCAAT/enhancer binding protein γ (C/EBPγ) is a ubiquitously expressed member of the C/EBP family of transcription factors that was originally described as an inhibitor of C/EBP transactivation potential. Previously, we reported that C/EBPγ augments the activity of C/EBPβ in lipopolysaccharide (LPS) induction of the interleukin-6 (IL-6) and IL-8 promoters in a B lymphoblast cell line. This ability of C/EBPγ to augment C/EBPβ transactivation potential is dependent upon its dimerization with C/EBPβ and is potentiated by coactivation of NF-κB. Here, we demonstrate a profound deficit in LPS-induced cytokine and chemokine expression in C/EBPγ-deficient mouse embryonic fibroblasts (MEFs) when compared to wild type (wt) MEFs. Chromatin immunoprecipitation analysis of two C/EBP target genes, IL-6 and IL-1β showed defective LPS-induced recruitment of C/EBPβ and C/EBPδ to the target promoters of these genes in C/EBPγ-deficient MEFs. These same promoters showed reduced LPS-induced NF-κB p65 occupancy. Furthermore, LPS-induced expression of several cytokine and chemokine genes was reduced in a tissue-specific manner in C/EBPγ-deficient mice when compared to wt mice. These findings demonstrate that C/EBPγ plays an important role in LPS-induced transcriptional activation of many cytokine and chemokine genes.