Docosahexaenoic acid (DHA) shows anti-inflammatory and/or anticancer effects in some cells. Activator protein-1 (AP-1) regulates cellular proliferation and apoptosis. Although recent studies demonstrate the association between gastric cancer risk and DHA, the exact molecular mechanism has not been clarified. We investigated whether AP-1 mediates DHA-induced apoptosis of gastric cancer cells. We found that DHA induced cell death and DNA fragmentation in parallel with the activation of extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK) as well as AP-1. DHA increased the protein levels of p53, cytochrome c, and Bax in gastric cancer cells. DHA-induced DNA fragmentation and protein levels of p53, cytochrome c, and Bax were inhibited in the cells transfected with c-jun dominant-negative mutant (TAM67). Because JNK and ERK are upstream signaling for AP-1 activation, we suggest that DHA-induced activation of AP-1 may mediate apoptosis of gastric cancer cells by inducing the expression of apoptotic genes in gastric cancer cells.