Hepatocellular carcinoma (HCC) remains one of the most predominant types of digestive system malignancies worldwide. TNF-related apoptosis-inducing ligand (TRAIL) is a biological cytokine with the mentioned specificity, but some tumor cells' resistance limits its use as a therapeutic approach. The present study aimed to investigate thymoquinone (TQ) and TRAIL's combined effect and the potential mechanisms in human hepatic HepG2 carcinoma cells.Cell viability and IC50 dose for TQ and TRAIL, alone and in combination, were determined using the MTT method. ELISA evaluated the expression levels of 8-Hydroxy-2'-deoxyguanosine. The apoptosis rate was assessed by flow cytometry, ELISA cell death assay, and caspase 8 activity assays. The mRNA and protein evaluation of candidate genes, including survivin, Bcl-2, XIAP, c-IAP1, c-IAP2, and c-FLIP, were accomplished before and after the treatment using qRT-PCR and Western blot analysis, respectively.Our results showed that TQ synergistically increased TRAIL's cell toxic effects as follows: TQ plus TRAIL > TRAIL > TQ. TQ could sensitize the HepG2 cells against the TRAIL-induced apoptosis and amplify the caspase 8 activity. This outcome is achieved by decreasing the mRNA and protein expression levels of anti-apoptotic genes.Our findings suggest that TQ can sensitize the human HCC cell line HepG2 against TRAIL by inducing the death receptor pathway. Moreover, these agents' combinational therapy might promise a therapeutic regimen for improving the clinical efficacy of TRAIL-induced apoptosis in patients with HCC.