In addition to their central role in energy metabolism, mitochondria can shape intracellular calcium (Ca2+) signaling through the concerted activity of Ca2+uptake to the matrix through the mitochondrial calcium uniporter, and efflux from the matrix mediated by the mitochondrial Na+/Ca2+exchanger, NCLX. Ca2+levels in the cytosol are known to regulate both metabolism and autophagic activity. Here, we describe a novel and direct relationship between NCLX, cytosolic Ca2+, and autophagic activity. We find that conditions that stimulate autophagyin vivoandin vitrosuch as caloric restriction and nutrient deprivation upregulate NCLX expression in hepatic tissue and cells. Conversely, siRNA-mediated knockdown of NCLX impairs basal and starvation-induced autophagy, by decreasing LC3 II flux. Similarly, acute inhibition of NCLX activity by CGP 37157 affects early steps of autophagy activation in hepatocyte-derived cells and mouse embryonic fibroblasts, resulting in reduced autophagic activity. Inhibition of NCLX caused decreased cytosolic Ca2+levels, and pre-incubation with intracellular Ca2+chelator BAPTA-AM promotes similar results to those of CGP 37157 on autophagy. BAPTA-AM did not exhibit any additive effect on NCLX inhibition of autophagy, demonstrating that mitochondrial Ca2+efflux regulates autophagy through the modulation of cytosolic Ca2+signaling. Collectively, our results demonstrate that the mitochondrial Ca2+extrusion pathway NCLX is an important regulatory node linking nutrient restriction and autophagy regulation through the modulation of cytosolic Ca2+levels.