Hypoxia‐inducible factors (HIFs) provoke adaptation to hypoxic stress occurring in rapidly growing tumor tissues. Therefore, overexpression of HIF‐1 or HIF‐2 is a common feature in hepatocellular carcinoma but their specific function is still controversially discussed. To analyze HIF function in hypoxia‐induced cell death we created a stable knockdown of HIF‐1α and HIF‐2α in HepG2 cells and generated tumor spheroids as an in vitro hepatocellular carcinoma model. Knockdown of HIF‐1α enhanced expression of HIF‐2α and vice versa. Unexpectedly, knockdown of HIF‐1α or HIF‐2α increased cell viability as well as spheroid size and decreased caspase‐3 activity. Antiapoptotic Bcl‐X_L expression increased in both knockdown spheroids, whereas proapoptotic Bax was only reduced in HIF‐1α‐knockdown cells. Furthermore, an HIF‐2α‐knockdown significantly increased Bcl‐2/adenovirus E1B 19 kDa‐interacting protein 3 (BNIP3) expression in an HIF‐1α‐dependent manner. Concomitantly, electron microscopy revealed a substantial increase in autophagosomal structures in HIF‐2α‐knockdown spheroids and mito‐/lysotracker costaining confirmed lysosomal activity of these autophagosomes. Blocking autophagosome maturation using 3‐methyladenine restored cell death in HIF‐2α‐knockdown clones comparable to wildtype cells. Conclusion: An HIF‐1α‐knockdown increases HIF‐2α expression and shifts the balance of Bcl‐2 family members toward survival. The knockdown of HIF‐2α raises autophagic activity and attenuates apoptosis by enhancing HIF‐1α expression. Our data indicate that enhanced expression of one HIF‐isoform causes a survival advantage in hepatocellular carcinoma development. HEPATOLOGY 2010