Objective: This study aims to explore the role and mechanism of cardiolipin acyltransferase 1(ALCAT1) gene deficiency in Kirsten rat sarcoma viral oncogene homolog(KRAS) induced lung adenocarcinoma. Methods: Doxycycline diet induced lung adenocarcinoma in CC10rtta-KRAS/ALCAT1 KO mice and CC10rtta-KRAS mice. Lung tumors in mice were detected by micro computed tomography (micro-CT) and observed by HE pathological section. A549 cells were divided into normal group, normal+Jenu group, hypoxia group and hypoxia+Jenu group. CoCl2 induced hypoxia and Jenuglitapin (Jenu) pharmacologically inhibited ALCAT1 enzyme. ROS levels were measured by reactive oxygen species (ROS) kits. Lactate concentration were measured by lactate kits. The mRNA and protein levels of related genes were detected by RT-PCR and Western blot in tissues and cells. Results: Compared with that CC10rtta-KRAS mice, the volume of lung tumors were reduced in CC10rtta-KRAS/ALCAT1 KO mice, when doxycycline diet for 8 weeks or 12 weeks. We observed more focal adenocarcinoma in CC10rtta-KRAS mice, while ALCAT1 deficiency decreased the expansion of lung adenocarcinoma lesions. Moreover, ALCAT1 deficiency reduced Akt phosphorylation levels in the doxycycline diet group. Pharmacological inhibition of ALCAT1 significantly decreased the contents of ROS and lactate, reduced Akt phosphorylation levels under hypoxic conditions in A549 cells. In addition, pharmacological inhibition of ALCAT1 suppressed HIF1α expression by preventing ROS production under hypoxia conditions in A549 cells. Conclusion:ALCAT1 deficiency prevented the development of lung adenocarcinoma. Pharmacological inhibition of ALCAT1 reduced HIF1α expression by preventing ROS generation, thereby inhibited glycolytic metabolites and affected Akt phosphorylation levels.