Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver disease. NAFLD can progress to further irreversible liver failure such as non-alcoholic steatohepatitis (NASH)-fibrosis and cirrhosis. However, specific regulator of NASH-fibrosis has not been established yet. Here, we found that glutathione peroxidase 7 (GPx7) was markedly expressed in NASH-fibrosis. Although GPx7 is known as antioxidant enzyme to protect other organs, whether GPx7 plays a role in NASH-fibrosis has not been studied yet. We found that knockdown of GPx7 in transforming growth factor-β (TGF-β) and free fatty acids (FFA)-treated LX-2 cells elevated expression of pro-fibrotic and pro-inflammatory genes and collagen synthesis. Consistently, GPx7 overexpression in LX-2 cells led to the suppression of ROS production and reduced expression of pro-fibrotic and pro-inflammatory genes. Moreover, choline-deficient amino acid defined, high fat diet (CDAHFD) feeding-induced NASH-fibrosis was significantly accelerated by knockdown of GPx7, as evidenced by up-regulated liver fibrosis and inflammation compared to CDAHFD control mice. Collectively, these results suggest that GPx7 might be a novel therapeutic target to prevent the progression and development of NAFLD.