The present study utilized non-targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) to investigate the metabolomic responses of sea cucumber (Apostichopus japonicus) juveniles under predation stress induced by sea stars (Asterina pectinifera) at various time points (3 h, 12 h, 72 h, and 96 h). The findings revealed significant temporal changes in the metabolic profiles of the sea cucumber juveniles under predation stress, with 25, 72, 55, and 53 metabolic products exhibiting significantly different expression levels at each time point (positive ion mode, P < 0.05), respectively. Notably, the impact of predation stress was most pronounced at the 12-h mark. Multivariate statistical methods, including principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA), further confirmed distinct clustering of the experimental group away from the control group at each time point, with the most pronounced separation occurring at 12 h, indicating a significant and time-dependent metabolic response to predation stress. Key metabolic pathways associated with predation stress were identified, such as carbon metabolism, pentose phosphate pathway, purine metabolism, riboflavin metabolism, longevity regulation, and antifolate resistance pathways, by integrating variable importance in the projection (VIP), fold change (FC), and P-value. KEGG enrichment analysis highlighted significant expression changes of key metabolites like carbamoyl phosphate, gluconolactone, inosine, 2'-deoxyguanosine, and adenylate in response to predation stress, potentially related to energy metabolism, antioxidant defense, signal transduction, and cellular stress responses. The study provides novel insights into the metabolic adaptability of sea cucumber juveniles to predation stress.