pH homeostasis is essential for development, proliferation and apoptosis of cells. Once the pH balances are broken, cell functions and survival will be affected. Nevertheless, moderate acidosis could result in adaptive responses for cell survival and increase tolerance to harmful stress. Here we found that acidic preconditioning (APC) could significantly increase the survival rate of Daphnia pulex, a freshwater invertebrate, during severe hypoxic insult. Meanwhile, the acidic treatment significantly increased the gene expression of hypoxia inducible factor (HIF). Both echinomycin, an inhibitor of HIF, and compound C, an inhibitor of AMP-activated protein kinase (AMPK), could effectively eliminate the acid-induced hypoxic tolerance and the enhanced transcription of HIF. Temsirolimus, an inhibitor of mammalian Target of Rapamycin (mTOR), though effectively abolished the increased transcription of HIF, improved the APC-mediated protection. This result suggests that the involvement of the HIF and AMPK and mTOR could signal the pathways in APC-induced protection against hypoxic insult.