Gemcitabine is a promising compound for the treatment of human lung cancer. Although apoptosis has been shown to play a role in certain cell types with gemcitabine, the steps leading to cell death after the drug-target interaction are not well understood. We studied the molecular mechanisms of gemcitabine-induced apoptosis and determined the role of p53 function on the cytotoxic effects of gemcitabine in human nonsmall cell lung cancer (NSCLC) H1299 and H1299/p53 cells. Here, we found that gemcitabine induced an apoptotic cell death via a Bcl-2-dependent caspase-9 activation pathway. Moreover, phosphorylated activation of extracellular signal-regulated kinase (ERK) was observed upon gemcitabine treatment. Genetical or pharmacological inhibition of ERK activation markedly blocked gemcitabine-induced cell death. Furthermore, inactivation of Akt was also involved in this event. Taken together, our observations indicate that ERK activation and Akt inactivation mediated gemcitabine-induced apoptosis independently of p53 in human NSCLC H1299 cells.