Afatinib is used to treat non-small cell lung cancer cells (NSCLC), and its mechanism involves irreversible inhibition of epidermal growth factor receptor (EGFR) tyrosine kinase. In this study, we examined if afatinib had cytotoxic action against NSCLC other than inhibition of tyrosine kinase. Afatinib (1–30 μM) caused apoptotic death in A549 NSCLC in a concentration-dependent manner. Afatinib triggered Ca influx without causing Ca release, and the Ca influx was unaffected by sodium orthovanadate (SOV, an inhibitor of tyrosine phosphatase), suggesting that afatinib-triggered Ca response was unrelated to its inhibition of tyrosine kinase. Addition of afatinib also promoted Mn influx. Ca influx triggered by afatinib was resistant to SKF96365 and ruthenium red (two general blockers of TRP channels) and, unexpectedly, Ni (a non-specific Ca channel blocker). Afatinib caused an increase in mitochondrial Ca level, an initial mitochondrial hyperpolarization (4 h) and followed by mitochondrial potential collapse (24–48 h). Afatinib-induced cell death was slightly but significantly alleviated in low extracellular Ca condition or under pharmacological block of mitochondrial permeability transition pore (MPTP) opening by cyclosporin A. Therefore, in addition to tyrosine kinase inhibition as a major anti-cancer mechanism of afatinib, stimulation of an atypical Ca influx pathway, mitochondrial Ca overload, and potential collapse in part contribute to afatinib-induced cell death.