Hepatocellular carcinoma (HCC) is one of the most common tumors worldwide. The prognosis of HCC is poor and the only curative option is liver resection or transplantation. Gene therapy is an exciting approach because it offers the potential of correcting the underlying genetic defect. Chronic hepatitis C is a recognized risk factor for HCC. Our study aimed to evaluate the effect of antisense oligodeoxynucleotides (AS-ODNs) in hepatitis C-associated HCC on the growth of the tumor cells grown in vitro fluid culture. This study included 20 patients with hepatitis C-associated HCC. Tumor cells grown in vitro fluid culture were divided into two groups, group 1 to which AS-ODN was added and group II no AS-ODN was added and served as a control group. Comparison of cell viability in both groups was done using: colony formation in soft agar, assay of LDH in culture supernatant, Trypan blue exclusion test, MTT assay and cell cycle analysis. Our results showed that there was statistically highly significant inhibition of growth of the tumor cells in group I (with AS-ODN) compared to group II (control group). Conclusion: Our in vitro study showed that AS-ODN has a significant inhibitory effect on the growth of hepatitis C-associated HCC cells grown in fluid culture, and would provide a scientific base for the possibility of use of AS-ODN in the treatment of HCC in the future.