Hepatocellular carcinoma is one of the most common tumors worldwide. Activator protein 1 (AP-1) is a nuclear transcription factor, and its transactivation is required for transformation in several cell lines. However, no direct correlation between AP-1 activity and human hepatocellular transformation has been proved. Here we analyzed the role of AP-1 on the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced human hepatocellular transformation. TPA promoted the formation of anchorage-independent colonies, induced the AP-1 activity, and enhanced the DNA-binding ability of AP-1 in human hepatocytes. The phosphorylation of extracellular signal-regulated protein kinases (ERKs) was increased by TPA and the TPA-induced AP-1 activity was inhibited by PD98059, indicating that TPA-induced AP-1 activation was via ERK pathway. Moreover, retinoic acid and PD98059, which inhibited the AP-1 activity, abolished the TPA-induced transformation. Our findings indicated that AP-1 and ERKs activations were required for TPA-induced human hepatocellular transformation. These studies suggested that AP-1 could be the target for the development of antihepatocellular transformation agents.