<p>This study applied non-targeted metabolomics technology based on LC-MS and targeted analysis to investigate the metabolic composition and changes, color-related chemical components, and enzymatic activity differences in pickled leaf mustard throughout the entire natural fermentation process. The results of Spearman’s correlation analysis showed that acid-catalyzed chlorophyll degradation, carbonyl ammonia reaction, ascorbic acid oxidative degradation, cell membrane structure damage and decreased defense mechanisms of antioxidant enzymes (such as POD, PAL, and APX) played dominant roles in color changes of pickled leaf mustard during fermentation. According to the results of KEGG enrichment, we noted that the amino acids-related metabolic pathways including cyanoamino acid metabolism, phenylalanine metabolism, tyrosine metabolism, tryptophan metabolism, glutamate metabolism, glutathione metabolism, and nitrogen metabolism were enriched. Moreover, three important lipid metabolism pathways including alpha-linolenic acid metabolism, linoleic acid metabolism, and arachidonic acid metabolism, were enriched. We also noted the biosynthesis pathways of two types of alkaloids (indole and isoquinoline alkaloids). The contents of important intermediate metabolites involved in color-regulation had changed a lot, including porphyrinogen, γ-aminobutyric acid, tyramine, dopamine, indole-3-acetic acid, N-Acetyl serotonin, glycerophosphocholine, and traumatic acid. Besides, one phenolic compound, two terpenoid secondary metabolites, and one isoquinoline alkaloid were potentially strongly correlated with color changes of tissues. The composition and content of phenolic substrates changed significantly as the fermentation tended to mature. This study would provide new insights into the related physiological mechanisms of color-change during fermentation in natural fermented leaf mustard and a theoretical reference for the quality control strategy of fermented vegetables.</p> Graphical abstract <p></p>