Abstract: Objective To observe the effects of betaine on the Hcy concentration, SAM/SAH ratio , and the RNA expression of lipid metabolism related genes in steatotic hepatocytes. Methods A hepatocyte steatosis model was established using 60 mg/L oleic acid and HepG2 cells. Steatosis HepG2cells were cultured in medium containing 0, 2.5, 5, and 10 mmol/L betaine for 24 h, respectively. The control group was cultured in regular medium containing no oleic acid and betaine. There were 3 subunits of every group. Lipid droplets in hepatocytes were observed after oil red O-hematoxylin staining. TG concentration was determined by enzymic method. Hcy level and SAM/SAH ratio were detected by HPLC while the expression of APOB, MTP, and DGAT2 mRNA were detected by RT-PCR. Results Compared with the control group, the model cells had higher Hcy in the medium (P<0.05), and higher TG was observed in both cells and medium (P<0.01). But the cellular SAM/SAH (P<0.05) and APOB mRNA expression (P<0.01) was lower. Compared with the model group, the 2.5 and 10 mmol/L betaine groups had higher TG incells (all P<0.05), while the 5 mmol/L betaine group had higher TG in medium (P<0.01). The SAM/SAH ratio in 10 mmol/L betaine group and the APOB mRNA expression in 5 and 10 mmol/L betaine groups were increased compared with the model cells (all P<0.01). The DGAT2 mRNA expression was higher in betaine groups than both the control group and model group (all P<0.05). Conclusion Betaine can decrease Hcy concentration in the steatotic hepatocytes’ medium and increase SAM/SAH ratio steatotic hepatocytes. Betaine enhanced TG excretion by increasing the APOB mRNA expression. Simultaneously, betaine increased the DGAT2 mRNA expression, which might increase the synthesis of TG.