Malolactic fermentation, started by lactic acid bacteria, plays a crucial role in the production of high-quality wines. As global warming increases the ethanol content in wines, the success of malolactic fermentation depends on selecting ethanol-tolerant strains, especially for wines from increasingly warm climates. Lentilactobacillus hilgardii Q19 was isolated and characterized as an indigenous malolactic bacterium with higher ethanol tolerance properties. In this study, it was indicated that ethanol stress had significant effects on ATPase activity, antioxidant system, and cell membrane of L. hilgardii Q19 by measuring the physiological indicators under stress which include H+-ATPase, Na+/K+-ATPase, Ca2+/Mg2+-ATPase activity, glutathione content, superoxide dismutase (SOD) activity and intracellular reactive oxygen species (ROS) content. The main metabolic pathways involved in ethanol stress such as ATP-binding cassette (ABC) transporters, pentose phosphate pathway, phosphotransferase system, glutathione metabolic pathway and two-component systems were screened by transcriptome sequencing analysis. The functions of the pentose phosphate pathway, pyruvate metabolic pathway and glycerolipid metabolism under ethanol stress were verified by constructing the L. hilgardii Q19 ethanol stress related key genes gntK, pyk, and glpK overexpression vectors. The above findings may contribute to our understanding of the metabolic pathways and regulatory mechanisms of L. hilgardii Q19 in response to ethanol stress.