Widespread autotoxicity is an important obstacle to natural regeneration of many plants. The rare relict plantMetasequoia glyptostroboidesis a difficult to natural regeneration and is affected by litter allelopathy. However, little is known about the potential influence of autotoxic substances on different regeneration stages ofM. glyptostroboides.We identified multiple chemical compounds of aqueous extracts from fresh (recently accumulated) and natural litter (mixture of litter different phases of decomposition), to evaluate the autotoxic effects of the four most important detected compounds applied individually on seed germination and seedling growth ofM. glyptostroboides.Results found that the 28 chemical compounds were identified in the aqueous extracts ofM. glyptostroboideslitter. The Jaccard similarity coefficient of chemical compounds in aqueous extracts of fresh and natural litter ofM.glyptostroboidesreached 50%. The number of chemical compounds in fresh litter was 5.56% more than that in natural litter. Catechol, trifluoroacetamide, benzoic acid and D-(+)-arabitol significantly affected seed germination rate, seed germination index, vigor index, shoot length and main root length ofM. glyptostroboides.Specifically, benzoic acid had the strongest inhibitory effect, followed by catechol, trifluoroacetamide and D-(+)-arabitol. The autotoxic effect was concentration dependent, low concentrations were positive and neutral, high and extremely high concentrations were negative for all the chemical compounds. Moreover, catechol, trifluoroacetamide, benzoic acid and D-(+)-arabitol were autotoxic substances affecting the natural regeneration ofM. glyptostroboides,as well as strongly inhibited at the shoot growth stage. This study confirms that natural regeneration ofM. glyptostroboidesis restricted by a large amount of litter coverage under the forest, highlighting how the chemical compounds responsible for the autotoxic characteristics ofM. glyptostroboidesaffect the different regeneration stages ofM. glyptostroboides.