Glucose and cAMP reciprocally regulate expression of the L-type pyruvate kinase (L-PK) gene by controlling the formation of a complex containing the carbohydrate response element binding protein (ChREBP) and the coactivator CREB binding protein (CBP) on the L-PK promoter. However, the role of posttranslational histone modifications on the opposing effects of glucose and cAMP on the L-PK gene is unknown. Using the highly glucose-sensitive 832/13 rat insulinoma cell line, we demonstrated that glucose regulates acetylation and methylation of various histone residues at the L-PK gene promoter. These glucose-dependent histone modifications correlated with an increase in the recruitment and phosphorylation of RNA polymerase II (Pol II) on the L-PK gene promoter. Conversely, the cAMP agonist forskolin prevented glucose-mediated expression of the L-PK gene by decreasing the acetylation of histones H3 and H4 on the promoter, decreasing the methylation of H3-K4 on the coding region, and increasing the methylation of H3-K9 on the coding region. These changes induced by cAMP culminated with a decrease in the glucose-dependent recruitment of phosphorylated Pol II to the L-PK gene promoter. Furthermore, maneuvers that interfere with the glucose-dependent assembly of ChREBP and CBP on the L-PK promoter, such as increasing intracellular cAMP levels, overexpression of a dominant-negative form of ChREBP, and small-interfering-RNA-mediated suppression of CBP abundance, all altered the acetylation and methylation of histones on the L-PK promoter, which decreased Pol II recruitment and subsequently inhibited transcriptional activation of the L-PK gene. We conclude that the effects of glucose and cAMP are mediated in part by epigenetic modulation of histones.