Silver nanoparticles (AgNPs) are widely used for manufacturing products containing antibacterial agents, as well as food technologies such as edible films and food packaging. Routes of AgNPs exposure are principally derived by contacting with certain medical sprays, food, toothpaste, and purification products. Previously, we showed that AgNPs induce endoplasmic reticulum (ER) stress and promote apoptosis progression in SH-SY5Y cells; however, whether AgNP-induced ER stress is able to trigger autophagy in vivo and in vitro, and the role of autophagy in AgNP-induced cytotoxicity remain unclear. In the present study, we found that increased intracellular calcium (Ca²⁺) levels arising from AgNP-induced-ER stress resulted in activation of calmodulin-dependent protein kinase kinase β (CaMKKβ) and adenosine 5′-monophosphate-activated protein kinase (AMPK), which downregulated the level of mammalian target of rapamycin (mTOR) and upregulated Beclin-1 to activate autophagy in SH-SY5Y cells. Specifically, inhibition of autophagy by the addition of chloroquine (CQ) or silencing of Beclin-1 significantly enhanced the cytotoxicity of AgNPs, suggesting that autophagy plays a protective role in AgNP-induced cell apoptosis. Furthermore, we showed that oral administration of AgNPs for 28 continuous days induced ER stress-mediated apoptosis and autophagy in rats via activation of CaMKKβ and AMPK. In summary, this study is the first to report that AgNPs induce protective autophagy via a Ca²⁺/CaMKKβ/AMPK/mTOR pathway in vivo and in vitro. Therefore, public exposure to AgNPs should arouse concerns regarding environmental safety and human health.