The influence of changes in dietary habits, chemical treatments, variations in the activity of one-carbon enzyme gene modifications, and liver function impairment make hepatocellular carcinoma (HCC) patients prone to marginal methyl nutrient deficiency. Hepatocellular carcinoma has consistently ranked among the top five cancer incidence rates around the world, yet the issue of methyl nutrient deficiency still lacks systematic basic scientific assessment. This study aims to investigate whether the deficiency of methyl nutrients, specifically folate, choline, and betaine, induces the stem cell-like properties of hepatocellular carcinoma cells and promotes malignant progression, as well as to clarify the potential mechanisms involved. We found that human hepatocellular carcinoma cells (HepG2) cultured in a low methyl nutrient environment exhibited the following: (1) Methyl nutrient deficiency induced self-renewal cancer stem cell characteristics in hepatocellular carcinoma cells, forming tumor spheres on the seventh day, with a greater number and larger volume observed in environments with low folate and low choline. Upon isolating the tumor spheres and re-culturing, we found that the sphere cells induced by methyl nutrient deficiency (2) showed increased intracellular NADPH, accompanied by elevated lactate concentration in the microenvironment and changes in energy-related indicators of Warburg metabolism. At the same time, the expression of post-reprogramming related proteins Sirt1 and DNMT1 was altered. In summary, methyl nutrient deficiency may promote the malignant progression of hepatocellular carcinoma through both epigenetic modifications and energy conversion. National Science and Technology Council (NSTC112-2320-B-567-001). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.