The most common cause of cancer liver is hepatocellular carcinoma (HCC) and it is an important cause of cancer-related death all over the world (Crissien and Frenette, 2014). The pathogenesis of hepatocellular carcinoma is a multifactorial process involving different molecular and cellular events that lead to the progressive accumulation of molecular alterations at both genetic and epigenetic levels (Ho et al., 2016). Epigenetic modification means the presence of heritable states of gene expression without alteration in DNA sequences. Deregulated epigenetics modifications lead to affection of gene transcription, chromosomal stability, and cell differentiation participating in induction of carcinogenesis. Epigenetic alterations may be changes in particular DNA regions or of the histone proteins around which DNA is organized including the methylation, hydroxymethylation, or acetylation (or a combination of these), as well as non-coding RNAs regulation of gene expression (Wang et al., 2015).P16 is a cell cycle regulator and a tumor suppressor protein; hence, its suppression promotes tumor progression (Narimatsu et al., 2004). P16 binds to Cyclin Dependent 
Kinases 4 and 6 (CDK4/6), this leads to inhibition of its kinase activity and so preventing the phosphorylation of Retinoblastoma tumor suppressor gene (Rb) (Rayess et al., 2012). Silencing of P16 gene occurs by hyper methylation of its promoter that is a commonly observed event in HCC (Narimatsu et al., 2004).So the aim of this study was to determine the methylation status of P16 in HCC induced by DEN and phenobarbital in rat model.