Metaboloepigenetics, the interrelationship between metabolic function and epigenetic regulation, is increasingly recognised as a plausible mechanism underlying the developmental origins of adult health and disease. Significantly, suboptimal culture conditions during preimplantation embryo development impair embryonic metabolism and epigenetic regulation, influencing embryo viability and post-transfer developmental outcomes. Lactate availability has been shown to alter preimplantation mouse embryo metabolism accompanied by a reduced NAD+:NADH ratio. As NAD+ availability modulates the activity of histone deacetylases, lactate availability plausibly influences the preimplantation embryo epigenome. Therefore, this study aimed to determine whether altered lactate availability modified H3K9 and H3K27 acetylation (ac) levels. Pronucleate mouse embryos from superovulated females were cultured in G1/G2 in which the lactate:pyruvate (L:P) ratio was reduced (L:P=0) or increased (L:P=60) from control levels (L:P=30). Day 3 morula NADH autofluorescence, and day 3 morula and day 5 blastocyst H3K9ac and H3K27ac immunofluorescence were quantified, along with blastocyst total cell number A reduction in the L:P ratio did not alter total cell number, however it significantly increased endogenous NAD+ levels in morulae (P<0.01), decreased H3K9ac in both morulae (P<0.01) and blastocysts (P<0.01), and reduced H3K27ac in morulae (P<0.01). Conversely, increasing the L:P ratio had the opposing effect, inducing a significant decrease in endogenous NAD+ availability (P<0.05) and increasing H3K9ac levels at the morula stage (P<0.01). Increasing the L:P ratio however decreased H3K27ac levels in blastocysts (P<0.05). These data reveal that altered lactate availability profoundly affects endogenous NAD+ levels, accompanied by significant restructuring of the embryo acetylation landscape. These findings highlight the ability of culture media composition to affect metaboloepigenetic regulation within the preimplantation embryo.