Glucose is a principal metabolic fuel in the central nervous system, but, when glucose is unavailable, the brain can utilize alternative metabolic substrates such as monocarboxylates to sustain brain functions. This study examined whether the replacement of glucose with monocarboxylates (particularly pyruvate and lactate) had an equivalent effect of glucose on neuronal survival in rat hippocampal organotypic slice cultures, or ameliorate the neurotoxicity induced by amyloid β-peptide (Aβ). The possible mechanism was also explored. We found that pyruvate and lactate alone increased cell death in the hippocampal slice cultures at 24 and 48 h. Supplementation of glucose-containing culture media and Aβ-treated culture media with pyruvate, but not lactate, attenuated cell death as strong as with trolox, known as a reactive oxygen species scavenger, and niacinamide, an NAD+ precursor, and this protective effect was reversed by α-cyano-4-hydroxycinnamic acid. Pyruvate significantly increased the aconitase activity and the NAD+ levels in the hippocampal slices in the presence of Aβ, but did not maintain the ATP levels. Our results indicate that pyruvate and lactate alone cannot replace glucose as an alternative energy source to preserve the neuronal viability in the hippocampal slice cultures. Pyruvate, in the presence of glucose, improves neuronal survival in the hippocampal slice cultures and also protects neurons against Aβ-induced cell death in which mitochondrial NAD(P) redox status may play a central role.