The meadow soil in the Tibetan Plateau is highly susceptible to freeze-thaw changes under future climate conditions, and hence this study aims to elucidate the responses of meadow soil microbial activities to increasing number of freezing-thawing cycles. [Methods] A meadow soil was collected from the seasonal FT region of the Tibetan Plateau. Five different FT scenarios respectively with 1, 3, 5, 7 and 9 FT cycles (hereafter termed as C1, C3, C5, C7 and C9) were simulated and compared with the non-FT treatment (N9), to identify the varying responses of dissolved organic carbon (DOC), NO3-N, NH4-H, soil microbial biomass carbon (SMBC) and nitrogen (SMBN), as well as CO2 and N2O emission rates. [Results] 1) Compared with the non-FT N9, the SMBC was significantly decreased by 23.64% after C9, while the DOC significantly increased by 10.7%, cumulatively releasing 71.10% and 321.40% more CO2 and N2O; 2) With increasing number of FT cycles, the DOC and the activities of β-glucosidase and leucine aminopeptidase showed nonlinear patterns, which declined first to the lowest values after C5 and then gradually increased at C7 and C9. The CO2 emissions rates peaked after C2 (1.83 times of that from the non-FT soil), whilst the N2O emission rates roared up after C5 (reaching 8.01 ~ 25.43 times of that from the non-FT soil). 3) With the increasing number of FT cycles, the SMBN did not vary significantly, yet the NO3-N and acetylaminoglucosidase activities gradually increased, while the NH4-N steadily decreased. [Conclusion] The effects of multiple freeze-thaw cycles on soil microorganisms were not just repetitions of a single freeze-thaw event. After five freeze-thaw cycles in alpine meadow soils, the changes in pore structure and microbial deaths induced by soil water freezing and swelling had been basically stabilized, while the surviving microbial communities gradually adapted to the new temperature conditions and revived their activities in further freeze-thaw cycles. This casted a new light to help advance our current understanding as to how soil microbes overwinter in the seasonal freeze-thaw regions.