Background: Histone acetylation and deacetylation play an important role in transcription and gene expression. The acetylation status of histones and non-histone proteins is determined by histone acetyl-transferases (HATs) and histone deacetylases (HDACs). Histone deacetylase inhibitors (HDACIs) induce various molecular and extracellular effects, leading to potent anti-cancer activities. The current study was designed to investigate the effect of valproic acid )VPA( on extrinsic and intrinsic apoptotic pathways, cell viability, and apoptosis in hepatocellular carcinoma PLC/PRF5 cell line. Materials and Methods: The hepatocellular carcinoma PLC/PRF5 was cultured. When cells approximately became 80% confluent, 3×105 cells were seeded into 96 and 24‑ well plates. After 24 h, the medium was replaced with the medium contains VPA (except control groups which were treated with DMSO). After 24, 48, and 72 h, to determine cell viability, cell apoptosis and gene expression, MTT assay, flow cytometry and Real-time quantitative RT-PCR (qRT-PCR) were done respectively. Results: VPA inhibited cell viability, induced apoptosis, decreased Bcl-2, and Bcl-xL and increased DR4, DR5, FAS, FAS-L, TRAIL, BAX, BAK and APAF1 significantly. Conclusion: It seems that VPA can play its role through intrinsic and extrinsic apoptotic pathways in hepatocellular carcinoma PLC/PRF5 cell line.