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BACKGROUND Scrophularia ningpoensis is a famous medicinal crop and continuous cropping seriously affected the yield and quality, but little is known about the influence of continuous cropping on metabolic pathways. In this study, the difference of protein abundance between continuous cropping and non-continuous cropping of Scrophularia ningpoensis roots was studied by proteomics, and the molecular mechanism that resists Scrophularia ningpoensis from continuous cropping was explored. RESULTS The results suggested that continuous cropping in Scrophularia ningpoensis altered the expression of proteins related to starch and sucrose metabolism, glycolysis/gluconeogenesis, pentose phosphate pathway, TCA cycle, phenylalanine, tyrosine and tryptophan biosynthesis, phenylpropanoid biosynthesis, terpenoid backbone biosynthesis, monoterpenoid biosynthesis, sesquiterpenoid and triterpenoid biosynthesis, and steroid biosynthesis. Among these processes, the most affected were phenylpropanoid biosynthesis and starch and sucrose metabolism, which may be important for continuous cropping resistance. CONCLUSION In conclusion, the effect of continuous cropping on Scrophularia ningpoensis was demonstrated at the proteome level in this work, and identified candidate proteins that may cause continuous cropping reactions. The paper provides the theoretical foundation and scientific reference for enhancing the continuous cropping resistance of Scrophularia ningpoensis. This article is protected by copyright. All rights reserved.