Biotin status and function are decreased during oral carbamazepine (CBZ) administration in both humans and rats, but it is not known whether biotin supplementation can prevent these decreases. To test the effectiveness of pharmacologic biotin supplementation during CBZ administration, 55 rats were randomly divided into 4 groups (0.06 mg biotin/kg diet +/- 3.75 g CBZ/kg diet and 6.0 mg biotin/kg diet +/- 3.75 g CBZ/kg diet). CBZ and biotin-supplemented diets began on d 5 and 26, respectively, and continued through d 68. Rats (n = 5/group) were killed on d 5, 26, 47 or 68. CBZ reduced serum and liver free biotin (P < 0.05), whereas biotin supplementation during CBZ administration maintained biotin status. CBZ also decreased specific activities and abundance of biotinylated pyruvate and acetyl CoA carboxylases (PC and ACC, P < 0.05) in brain and liver, whereas biotin supplementation prevented these decreases for ACC. Specific activity of PC was maintained upon biotin supplementation, but the abundance of biotinylated PC remained significantly decreased. Brain and serum lactate were elevated after 68 d of CBZ treatment and were reduced to control lactate concentrations upon biotin supplementation (P < 0.05). Conversion of lactate to pyruvate and simultaneous generation of NADH during biotin supplementation could explain how increases in PC activity occur without changes in the abundance of biotinylated PC because we found NADH to be an activator of PC activity in vitro. These results support the use of biotin supplementation as a concurrent strategy during CBZ administration to help maintain biotin status, function of biotin-dependent enzymes and decrease CBZ-induced lactate accumulation.