Objective To explore the effect and potential mechanism of Guiqi Yiyuan ointment on rats injured by bystander effects of ¹²C⁶⁺ radiation. Methods A total of 70 Wistar male rats were randomized into the following groups (n=10):blank control group, radiation 24 h group, Guiqi Yiyuan ointment + radiation 24 h group, Guiqi Yiyuan ointment 24 h group,radiation 7 d group, Guiqi Yiyuan ointment + radiation 7 d group, and Guiqi Yiyuan ointment 7 d group. Fourteen days before irradiation, rats in the groups with Guiqi Yiyuan ointment treatment received the same volume and concentration of Guiqi Yiyuan ointment. Other rats received the same amount of normal saline. After 14 days, the right lungs of each radiation group and the Guiqi Yiyuan ointment + radiation group were irradiated with a single 4 Gy ¹²C⁶⁺ beam (exposing only the right lung). The blank control group and each Guiqi Yiyuan ointment group were not irradiated. Rats in each group were sacrificed at different time points such as 24 h and 7 d, and lung tissues were collected. The histology of the left and right lungs of each group was examined by HE staining.The mRNA and protein expression levels of TGF-β₁ and p-Smad3 in the left and right lung tissues were quantified by Q-PCR and Western blotting, respectively. Results HE staining results showed the normal structure of the right lung alveoli of blank control group and each Guiqi Yiyuan ointment group. In radiation 24 h group, the alveolar septum of both lungs increased with inflammatory cells infiltrated. In Guiqi Yiyuan ointment + radiation 24 h group, both lungs showed some broken alveolar septum and some infiltrated inflammatory cells. In radiation 7 d group, the alveolar septal fissure was obvious, the alveolar cavity expanded irregularly,the inflammatory cells infiltrated, and the degree of infiltration in the left lung was lighter than that in the right lung. In Guiqi Yiyuan ointment + radiation 7 d group, there was no fibrous hyperplasia, no congestion in the alveolar cavity, and the degree of left lung injury was lighter than that of the right lung. Q-PCR and Western blotting results showed that compared with blank control group,the expression levels of TGF-β₁ mRNA and protein in the left and right lung tissues of rats in radiation 24 h group and radiation 7 d group increased (P<0.05), the expression level of p-Smad3 protein increased (P<0.05), the expression level of Smad6 mRNA in the right lung decreased (P<0.05), and the expression level of Smad6 mRNA in the left lung increased (P<0.05), the expression of Smad6 protein decreased in the left and right lung tissues (P<0.05), and the expressions of TGF-β₁ mRNA and protein in radiation 7 d group were higher than those in radiation 24 h group (P<0.05), and there was a time effect. The expressions of TGF-β₁ mRNA and protein and p-Smad3 protein in the left and right lungs of rats in Guiqi Yiyuan ointment + radiation group decreased, while the expression of Smad6 mRNA and protein increased (P<0.05). Conclusion Guiqi Yiyuan ointment has a protective effect on rats with bystander effects of radiation. The mechanism may be related to the downregulation of TGF-β₁ and p-Smad3, but increased Smad6 in the left and right lung tissues.