Background <p>Nicotinamide riboside (NR) is an NAD⁺ precursor and is reported to upregulate NAD⁺ levels in several tissues. We previously demonstrated that NR exhibited protection against axon loss induced by tumor necrosis factor (TNF) with upregulation of SIRT1 and enhancement of autophagy. We recently found that NR upregulated phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK) protein in the optic nerve. The present study aimed to examine whether a SIRT1 inhibitor prevents axonal protection by NR and alters p-AMPK expression.</p> Methods and results <p>Rats were given intravitreal injection of a SIRT1 inhibitor prior to simultaneous injection of NR and TNF. The SIRT1 inhibitor alone or NR alone group was also made. SIRT1 and p-AMPK expression in optic nerve was investigated by immunoblot analysis. We examined the localization of SIRT1 and p-AMPK in the retina and optic nerve. Morphometric analysis showed axonal protection by NR in TNF-induced axon loss, and this protective effect was significantly prevented by the SIRT1 inhibitor. The SIRT1 inhibitor alone did not alter axon numbers significantly compared with the control group. The SIRT1 inhibitor significantly decreased p-AMPK levels in optic nerves in NR plus TNF-treated eyes. On the other hand, the SIRT1 inhibitor alone did not alter p-AMPK levels. Co-localization of SIRT1 and p-AMPK was observed in the retina and optic nerves in NR-treated eyes.</p> Conclusions <p>SIRT1 exists upstream of AMPK, and axonal protection of NR is associated with the SIRT1-AMPK pathway.</p>