Understanding tumor‐specific metabolism under hypoxia is important to find novel targets for antitumor drug design. Here we found that tumor cells expressed higher levels of cytosolic acetyl‐CoA synthetase (ACSS2) under hypoxia than normoxia. Knockdown of ACSS2 by RNA interference (RNAi) in tumor cells enhanced tumor cell death under long‐term hypoxia in vitro. Our data also demonstrated that the ACSS2 suppression slowed tumor growth in vivo. These findings showed that ACSS2 plays a significant role in tumor cell survival under hypoxia and that ACSS2 would be a potential target for tumor treatment. Furthermore, we found that tumor cells excreted acetate and the quantity increased under hypoxia: the pattern of acetate excretion followed the expression pattern of ACSS2. Additionally, the ACSS2 knockdown led to a corresponding reduction in the acetate excretion in tumor cells. These results mean that ACSS2 can conduct the reverse reaction from acetyl‐CoA to acetate in tumor cells, which indicates that ACSS2 is a bi‐directional enzyme in tumor cells and that ACSS2 might play a buffering role in tumor acetyl‐CoA/acetate metabolism. (Cancer Sci 2009; 100: 821–827)