The G-protein Gαi2 mediates signaling in a variety of processes. Induced expression of Gαi2 by butyrate and various transcription factors has been established, but transcriptional suppression has not previously been explored. Using HepG2 and K562 cells in culture, we show here that whereas both C/EBPα and C/EBPβ induced transcription from the Gαi2 gene promoter, C/EBPα, but not C/EBPβ, inhibited butyrate-induced Gαi2 expression. Because the transcriptional effect of butyrate on this gene promoter is largely mediated by the transcription factor Sp1, we investigated whether C/EBPα influenced Sp1-induced Gαi2 gene transcription. Binding of C/EBPα to a C/EBP response element in Gαi2 gene promoter inhibited Sp1-induced promoter activity. ChIP analysis showed decreased butyrate-induced recruitment of Sp1 to the Gαi2 gene promoter in response to C/EBPα treatment. Incubating cells with acetate or transfecting them with expression plasmid for either the acetyltransferase p300 or CREB-binding protein (CBP) reversed the antagonistic effect of C/EBPα on Sp1-dependent gene transcription, suggesting that the mechanistic basis for the antagonism is related to the squelching of co-activator acetyltransferase(s) by C/EBPα or the acetylation of Sp1 and/or C/EBPα. This work reveals that C/EBPα plays a dual role as an activator and as a repressor of Gαi2 gene transcription.