Safety concerns have been raised with regard to silver nanowires (AgNWs) because of their extensive applications. Recently, ultralong AgNWs have shown physical properties superior to those of short AgNWs. However, little is known about their toxicity and potential risks. In this study, we demonstrated a series of ultralong AgNWs-induced biological effects in human lung cancer epithelial cells (A549). Ultralong AgNWs treatments induced ROS generation, mitochondria-mediated apoptosis, and self-protective autophagy at nonlethal concentrations. In contrast to some previous reports, apoptosis was found not to correlate with the reduction of intracellular ROS. Measuring the processing of ROS generation, apoptosis and autophagy, we demonstrated that ROS not only enhance mitochondrial damage, but also raise protective autophagic flux in ultralong AgNW-treated cells. Moreover, ultralong AgNWs were found to be internalized into the cytoplasm of the epithelial cells. This study not only investigates ultralong AgNWs-induced cytotoxicity but also pinpoints ROS as a key signal in mechanisms of their toxicity.