Knockdown of endothelin A receptor expression inhibits osteosarcoma pulmonary metastasis in an orthotopic xenograft mouse model. Previous in vitro studies suggest that the endothelin A receptor (ETAR) could be a potential therapeutic target for osteosarcoma (OS) metastasis. In the present study, we assessed for the first time the role of ETAR in OS proliferation and pulmonary metastasis in vivo. MG-63 human OS cells were stably transduced with ETAR shRNA or scramble control shRNA and injected into the tibia of nude mice to generate an orthotopic xenograft OS model. The mice were divided into three groups (n=10 each group): i) the untransduced control group, where the mice were injected with untransduced MG-63 cells; ii) the scramble control group, where the mice were injected with cells stably transduced with the scramble control shRNA; iii) the ETAR-shRNA group, where the mice were injected with cells stably transduced with ETAR shRNA. The ETAR shRNA knocked down more than 75% of endogenous ETAR expression and significantly inhibited invasion, but not proliferation/viability of MG-63 cells in vitro. In the orthotopic xenograft OS mouse model, no significant difference in the tumor volume was observed over time among the untransduced control, the scramble control and the ETAR-shRNA groups. However, a combination of clinical signs, organ examinations and quantitative clonogenic lung metastasis assays showed that lung metastasis in the ETAR-shRNA group was significantly lower than that in the control groups. Immunohistochemical analyses revealed that the matrix metalloproteinase-2 (MMP-2) expression was significantly reduced in the ETAR-shRNA group compared with the control groups. The results were confirmed with western blot analyses using primary tumor tissues or the stably transduced MG-63 cells. In conclusion, we demonstrated in an orthotopic xenograft OS model that ETAR is critical for OS pulmonary metastasis, but not for tumor growth. This study provides the first in vivo evidence suggesting an important role for ETAR in OS pulmonary metastasis.