Active autophagy coupled with rapid mitochondrial fusion and fission constitutes an important mitochondrial quality control mechanism and is critical to cellular health. In our previous studies, we found that exposure of cells to nicotinamide causes a decrease in mitochondrial content and an increase in mitochondrial membrane potential (MMP) by activating autophagy and inducing mitochondrial fragmentation. Here, we present evidence to show that the effect of nicotinamide is mediated through an increase of the [NAD(+)]/[NADH] ratio and the activation of SIRT1, an NAD(+)-dependent deacetylase that plays a role in autophagy flux. The [NAD(+)]/[NADH] ratio was inversely correlated with the mitochondrial content, and an increase in the ratio by the mobilization of the malate-aspartate shuttle resulted in autophagy activation and mitochondrial transformation from lengthy filaments to short dots. Furthermore, treatment of cells with SIRT1 activators, fisetin or SRT1720, induced similar changes in the mitochondrial content. Importantly, the activators induced mitochondrial fragmentation only when SIRT1 expression was intact. Meanwhile, MMP did not increase when the cells were treated with the activators, suggesting that the change in MMP is not induced by the mitochondrial turnover per se and that elevation of the [NAD(+)]/[NADH] ratio may activate additional mechanisms that cause MMP augmentation. Together, our results indicate that a metabolic state resulting in an elevated [NAD(+)]/[NADH] ratio can modulate mitochondrial quantity and quality via pathways that may include SIRT1-mediated mitochondrial autophagy.