Ophiopogon japonicus (Thunb.) Ker Gawl. (OJ), a well-known Chinese herbal medicine, possess anticancer effects in various type of cancers, particularly in lung cancer. However, the underlying mechanisms remain unclear. In present study, the effects of OJ against NCI-H1299 human lung cancer cells were investigated, and the underlying mechanisms were explored using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF-MS)-based cell metabolomics. As a result, OJ inhibited the proliferation, induced the apoptosis and suppressed the migration of NCI-H1299 cells. A total of 22 differential metabolites responsible for the effects of OJ were screened and annotated based on the LC-MS-based cell metabolomics approach. The altered metabolites were involved in 3 metabolic pathways, including glycerophospholipid metabolism, ether lipid metabolism and glutathione metabolism. These results showed that cell metabolomics-based strategies are promising tools to discover the action mechanisms of OJ against lung cancer cells. This study was supported by the funding grants from University of Macau (File no. MYRG2019-00121-ICMS and MYRG2018-00207-ICMS), the National Natural Science Foundation of China (File no. 82104354), Science and Technology Development Fund, Macau SAR (File no. 0016/2021/A), Guangdong Basic and Applied Basic Research Foundation (File no. 2022A1515012154), and the Shenzhen-Hong Kong-Macau S&T Program (Category C) (SGDX2020110309420200).