<p>Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. Despite systemic and molecularly targeted therapies commonly used in HCC, resistance gained against drugs highly limits treatment efficacy. For many years, the effectiveness of animal venoms and natural compounds in cancer treatment has been investigated with increasing interest. Theranekron D6®, an alcoholic extract of <i>Tarantula cubensis</i> venom, is used for various purposes in the veterinary field. Recent studies focusing on the effects of theranekron on human tumors have intensively investigated its mode of action. However, the anti-cancer effect of theranekron in HCC has not been studied yet. Herein, we investigated the possible anti-cancer effects of theranekron in hepatocellular carcinoma HepG2 cells by examining the ERAD, UPR signaling and autophagy mechanisms. Also, we tested its effect on the expression level of PARP-1, cyclin proteins and VEGF-A by immunoblotting. Lastly, to further analyze its effect on tumorigenic features of HepG2 cells, we carried out the functional analysis. We also examined the effect of theranekron on cellular morphological characteristics. Our findings revealed that theranekron has strong anti-cancer properties on HepG2 cells by differently modulating the autophagy, ERAD and UPR signaling. Moreover, it markedly induced the cleavage of PARP-1 protein and reduced the levels of cyclins and VEGF-A. More strikingly, it strongly suppressed the tumorigenic features of HepG2 cells. Our data indicated that theranekron has significant anti-cancer effects on HepG2 cells. However, further in vitro and in vivo analysis is needed for theranekron to find a place in clinical applications.</p> Graphical Abstract <p>Mode of action of theranekron on HepG2 cells.</p>