Abstract: 【Objective】The purpose of this study was to clarify the effect of hydrogen sulfide(H₂S)on the accumulation of polyamines and secondary metabolites in birch suspension cultures. 【Methods】Different concentrations of H₂S donor sodium hydrosulfide(NaHS)were added to a birch suspension culture. High performance liquid chromatography and colorimetric assays were used to analyze the levels of different forms of polyamines, enzyme activity of arginine decarboxylase(ADC)and polyamine oxidase(PAO), and the levels of flavonoids, polyphenols and triterpenoids. 【Result】 The total polyamine content of birch suspension cultures increased initially and then decreased after NaHS treatment. Among the cells, the highest level of putrescine and spermidine was at 1 mmol/L NaHS after 24 h treatment, with increasing rates of 30.51% and 12.37% compared to the control, respectively. With the exception of perchloric acid(PCA)-insoluble spermidine conjugate, the content of free-, PCA-soluble and PCA-insoluble putrescines and conjugates increased after NaHS treatment. Similarly, NaHS treatment enhanced the activity of ADC and PAO, with increasing ranges of 15.91%-26.33% and 137.5%-250.12%, respectively. Treatment on birch suspension cell with 1 mmol/L NaHS and different concentrations of putrescine, spermidine, and spermine increased the polyphenol, flavonoid and triterpenoid levels, respectively. Polyphenol, flavonoid and triterpenoid reached maximum levels after combined treatment with 5 mmol/L putrescine and NaHS; the levels increased by 26.69%, 13.22% and 4.55%, compared to NaHS treatment, respectively. 【Conclusion】 These results indicate that H₂S treatment increased the levels of polyamines and secondary metabolites, and the combined treatment with H₂S and polyamines further enhanced the accumulation of secondary metabolites in birch suspension cultures.