Annexin A2 (ANXA2) overexpression is required for cancer cell proliferation; however, the molecular mechanisms underlying ANXA2-mediated regulation of the cell cycle are still unknown. ANXA2 is highly expressed in non-small cell lung cancer (NSCLC) and is positively correlated with a poor prognosis. NSCLC A549 cells lacking ANXA2 exhibited defects in tumor growth in vivo and in cell proliferation in vitro without cytotoxicity. ANXA2 knockdown induced cell cycle arrest at G(2) phase. Unexpectedly, ANXA2 silencing increased the expression of p53 and its downstream genes, which resulted in p53-dependent and -independent G(2) arrest. Aberrant JNK inactivation, which was observed in ANXA2-deficient cells, inhibited cell proliferation following G(2) arrest. A lack of ANXA2 caused a loss of JNK-regulated c-Jun expression, resulting in an increase in p53 transcription. These results demonstrate a novel role for ANXA2 in NSCLC cell proliferation by facilitating the cell cycle partly through the regulation of p53 via JNK/c-Jun.