Background: Vitamin D, especially its most active metabolite 1,25-dihydroxyvitamin D₃(Vit D) is essential in regulating a wide variety of biologic processes, such as regulating mesangial cell activation. The objective of this study was to assess the histopathological changes of effectiveness of Vit D as a protective agent against radiation induced lung injury. Materials and Methods: Eighteen Wistar rats were divided into three groups: control group (group 1:4 rats), irradiation alone group (group 2:7 rats) and irradiation+vit D (group 3:7 rats). Rats in group 2 and 3 were exposed to 20 Gy radiations to the right lung in a Co⁶⁰ radiotherapy machine under general anesthesia. Additionally, rats in group 3 received Vit D at a single dose of 0.2 mcg injected IM 2 hours before exposure to irradiation. Rats were sacrificed and lungs were dissected fifty days after post-irradiation. Myofibroblasts and vitamin D₃receptors (VDR) in extracted lungs were stained by immunohistochemistry using alpha-smooth muscle actin (SMA) and VDR antibodies. Blinded histological evaluation was performed to assess lung injury. Lung injury was assessed by the acute lung injury score and myofibroblastic differentiation score. Results: Acute lung injury scores and myofibroblastic differentiation scores were significantly lower in the radiation+vit D group compared to irradiation alone group (p=0.001 and p=0.001, respectively). Conclusion: This study indicates that administration of vit D plays a protective role against acute lung injury through blocking myofibroblastic differentiation.