Abstract EphA2 kinase is highly expressed in normal mammalian liver, but its functions are poorly defined. Here we show that EphA2 expression was down-regulated in majority of human hepatocellular carcinoma (HCC) from three different populations. In mice, EphA2 deletion led to enhanced susceptibility to hepatocarcinogenesis induced by diethylnitrosamine (DEN) in tumor multiplicity and burden compared with wild type mice. Acute DEN treatment induced EphA2 expression upregulation, concomitant with mitotic arrest in wild type liver. EphA2-null liver displayed significantly increased apoptosis compared with wild type. Further, acute DEN treatment caused substantially elevated Akt and ERK1/2 kinase activities in EphA2-null but not in wild type liver. Ephrin-A1 stimulation inhibited AKT and ERK1/2 and suppressed cell proliferation in vitro. Re-expression of EphA2 fully restored its function. Liver development displayed stable EphA2 expression and kinase activation increase by age coinciding with upregulated ephrin-A1. Human HCC and the mouse model show that EphA2 suppresses hepatocarcinogenesis by facilitating mitotic arrest in response to carcinogenic insults; inhibition of Akt and ERK is one possible mechanism involved in tumor progression. Together, these finding provide a mechanistic link between EphA2 and liver tumor development. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5009.