Increased hepatic iron deposition participate in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Hepcidin is a master iron regulator that decrease iron efflux from hepatocytes and its level in NAFLD is still controversial. 1,25-dihydroxyvitamin D3 (vitamin D3) is a potent regulator of hepcidin and its deficiency was linked with increased severity of NAFLD. Therefore, this study aimed to assess the effect of vitamin D3 on hepatic iron deposition and circulating hepcidin levels in NAFLD model induced in adult male albino rats. Materials and Method: Sixty-four adult male rats average age five months weighing 180-200 g were distributed to eight groups. Group (1): ND (normal diet) fed for 4 weeks, Group (2): ND+VD (normal diet with vitamin D3; injected twice weekly with vitamin D3; 5 μg/kg BW) fed for 4 weeks, Group (3): HFD (high- fat diet) fed for 4 weeks, Group (4): HFD+VD (high-fat diet with vitamin D3; injected twice weekly with vitamin D3; 5 μg/kg BW) fed for 4 weeks. Group (5): ND (normal diet) fed for 12 weeks, Group (6): ND+VD (normal diet with vitamin D3; injected twice weekly with vitamin D3; 5 μg/kg BW) fed for 12 weeks, Group (7): HFD (high- fat diet) fed for 12 weeks, Group (8): HFD+VD (high-fat diet with vitamin D3; injected twice weekly with vitamin D3; 5 μg/kg BW) fed for 12 weeks. Body mass index (BMI), abdominal circumference (AC), lipid profile, and serum levels of liver enzymes, hepcidin, IL-6, iron and ferritin, hepatic levels of iron and reactive oxygen species (ROS) were measured in all groups. Histology of hepatic tissues was examined using hematoxylin & eosin, Prussian blue, and Masson's trichrome stains. Results: Vitamin D3 induced significant reduction in BMI, AC, lipid profile parameters (except for high-density lipoprotein HDL which was increased), liver enzymes, hepcidin, IL-6, ferritin, hepatic iron and ROS., Whereas, significant increase in serum iron levels in 4 and 12W-HFD groups was noticed compared to their time-matched HFD groups. Additionally, vitamin D3 abolished the pathological changes associated with HFD in 4-week group and markedly attenuated the changes in 12-week group, and significantly diminished hepatic iron deposition and hepatic fibrosis in these groups in comparison to their time-matched HFD groups. Conclusion: Vitamin D3 protects against HFD-induced NAFLD in adult male albino rats by suppression of hepcidin level and subsequent reduction in hepatic iron deposition that decreased oxidative stress-mediated hepatic injury and fibrosis.