Hypoxia is a common feature of tumor microenvironment (TME), which promotes tumor progression, metastasis and therapeutic drug resistance via a myriad of cell activities in tumor and stroma cells. While targeting hypoxic TME is emerging as a promising strategy for treating solid tumors, preclinical development of this approach is lacking in the study of hepatocellular carcinoma (HCC). From a genome-wide CRISPR/Cas9 gene knockout screening, we identified aldolase A (ALDOA), a key enzyme in glycolysis and gluconeogenesis, as an essential driver for HCC cell growth under hypoxia. Knockdown of ALDOA in HCC cells leads to lactate depletion, and consequently inhibits tumor growth. Supplementation of lactate partly rescues the inhibitory effects mediated by ALDOA knockdown. Upon hypoxia, ALDOA is induced by HIF-1α and FTO-mediated m6A modification through transcriptional and post-transcriptional regulation, respectively. Analysis of TCGA shows that elevated levels of ALDOA are significantly correlated with poor prognosis of HCC patients. In a screen of FDA-approved drugs based on structured hierarchical virtual platforms, we identify sulfamonomethoxine derivative cpd-5 as a potential inhibitor to target ALDOA, evidenced by the anti-tumor activity of cpd-5 in preclinical patient-derived xenograft models of HCC. CONCLUSION: Our work identifies ALDOA as an essential driver for HCC cell growth under hypoxia, and we demonstrate that inhibition of ALDOA in hypoxic TME is a promising therapeutic strategy for treating HCC.This article is protected by copyright. All rights reserved.