Dynamic alterations in mitochondrial Ca2+ levels are recognized for their role in regulating both mitochondrial and cellular functions, as well as influencing energy metabolism (1). Recent studies reported that mitochondrial Na+/Ca2+ exchanger (NCLX) is regulated by PKA phosphorylation on its regulatory domain and PDE2 plays a crucial role as a physiological determinant in the PKA-mediated regulation of NCLX (2, 3). A major unsolved question is what links the cell membrane signaling to mitochondria NCLX regulation. Here, we fluorescently monitored [Ca2+]m efflux mediated by the NCLX in mice hippocampal neurons after excitotoxic insults. Interestingly, we found that nitric oxide (NO) is involved in the regulation of mitochondrial Ca2+ homeostasis since the inhibition of nitric oxide synthase (NOS) with L-NAME upregulated mitochondrial Ca2+ efflux and restored mitochondrial membrane potential after both glutamate and NMDA excitotoxic insults. Similar results were observed using the selective neuronal NOS inhibitor indicating that this effect is mediated by neuronal NOS. Moreover, NOS inhibition increased the level of NCLX phosphorylation at its PKA site. Interestingly, we observed that the regulative effects mediated by NOS inhibitors are completely lost in NCLX knockout mice, revealing that the regulation of mitochondrial Ca2+ mediated by NOS is NCLX dependent and that NOS acts through PDE2 to control NCLX. In conclusion, our results reveal that NO might serve as the link between cell membrane signaling and NCLX regulation. J Physiol Sci. 2015 Jan;65(1):11-24.Cell Rep. 2015 Oct 13;13(2):376-86.Cell Rep. 2022 Dec 6;41(10):111772.