Background/Objective. Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most prevalent chronic liver condition often linked to metabolic disor-ders such as obesity, type 2 diabetes, and dyslipidemia. MASLD can progress to more severe conditions like metabolic dysfunction-associated steatohepatitis, cirrhosis, and hepatocellular carcinoma. While lifestyle changes are currently the main therapeutic strategy, emerging evidence suggests that dietary micronutrients, such as vitamin B6 (VB6), could play an important role in the development and progression of MASLD through their involvement in one-carbon (1C) metabolism. Methods: This study inves-tigated the impact of dietary VB6 levels on the progression of MASLD using a modified high-fat diet (HFD)-induced mouse model. Thirty-two male C57BL/6J mice were ran-domly assigned to either an AIN-93G-based control diet or an HFD containing varying levels of dietary VB6 for eight weeks. At the end of the dietary intervention, physical parameters, clinical markers, hepatic and fecal metabolites, and gut morphology were evaluated. Results: Our findings demonstrated that dietary VB6 deficiency significantly affected hepatic B6 levels, lipid accumulation, and key metabolites involved in the 1C metabolic pathway. Notably, HFD-fed animals receiving low VB6 levels exhibited re-duced plasma triglyceride levels, increased hepatic triglyceride content, and alterations in homocysteine and glycine levels, suggesting disruptions in both lipid and 1C metab-olism. Interestingly, fecal glycerol and 3-hydroxyisovalerate levels were elevated in HFD-fed groups compared with those in the control group. Conclusions: These findings suggest that dietary VB6 plays a crucial role in regulating MASLD progression, particu-larly in the context of diet-induced MASLD, providing insights into potential dietary interventions for disease management.