Backgrounds: Hepatocellular carcinoma (HCC), Primary liver cancer, is the fifth most common cancer in men. Histone deacetylation causes chromatin condensation resulting in gene silencing and tumorigenesis. These enzymes have become a novel target for the treatment of cancer. Histone deacetylase inhibitors (HDACIs) can reactivate tumor suppressor genes (TSGs) by inhibition of histone deacetylases (HDACs) activity leads to apoptosis induction in cancer cells. Further, these compounds can induce apoptosis through the intrinsic/mitochondrial pathway. Previously, we reported the effect of valproic acid (VPA) and trichostatin A (TSA) on TSGs p21 WAF1/CIP1 (p21), p27 Kip1 (p27), and p57 Kip2 (P57) and also HDAC1 in colon cancer. The present study was designed to investigate the effect of VPA on the class I histone deacetylase (HDAC) 1, 2 and 3, TSGs p21and p53, and intrinsic mitochondrial pathway, pro-(Bax, Bak, and Bim) and anti-(Bcl-2, Bcl-xL, and Mcl-1) apoptotic genes, viability, and apoptosis in HCC HepG2 cell line. Materials and Methods: The HepG2 cells were cultured and treated with VPA. To determine viability, apoptosis, and the relative expression level of the mentioned genes, MTT assay, cell apoptosis assay, and qRT-PCR were done respectively. Results: VPA downregulated class I histone deacetylase (HDAC) 1, 2, and 3, Bcl-2, Bcl-xL, and Mcl-1 and upregulated p21, p53, Bax, Bak, and Bim resulting in apoptosis induction. Conclusion: VPA can induce apoptosis via activation of the intrinsic mitochondrial apoptotic pathway and also epigenetic reactivation of p21 and p53 through inhibition of class I HDAC 1, 2 and 3, activity.