Freeze-thaw processes profoundly affect soil carbon storage by altering microbial communities. However, the response mechanisms of microbial community properties and their carbon sequestration potential to freeze-thaw patterns remain poorly understood, especially as climate warming intensifies freeze-thaw patterns in permafrost regions. In order to understand how freeze-thaw processes regulate microbial communities and carbon storage, we conducted in situ investigations during four periods of seasonal freeze-thaw processes, including thawing, completely thawed, freezing, and completely frozen periods in the surface of the active layer of alpine permafrost in the Qinghai-Tibet Plateau. We analyzed microbial community composition, beta diversity, assembly processes, co-occurrence networks, and their associations with water-soluble organic carbon density (WSOCD). Our results revealed that species turnover dominated microbial beta diversity across seasonal freeze-thaw processes. Stochastic processes dominated microbial assembly, with bacterial communities showing higher drift and fungal communities exhibiting greater dispersal limitation. Phylogenetic groups associated with assembly processes further structured microbial network clusters, and Cluster 1 was negatively correlated with WSOCD. Freeze-thaw strength regulated WSOCD by altering soil and microbial community properties, collectively explaining 37% of the observed variation. These findings provide new insights into microbially mediated carbon dynamics in alpine permafrost ecosystems, offering valuable perspectives for predicting carbon sequestration ability responses under intensified freeze-thaw patterns driven by future warming.IMPORTANCEUnderstanding how seasonal freeze-thaw processes regulate microbial communities and carbon storage is vital for elucidating their interlinked dynamics in permafrost ecosystems. While microbial mediation of carbon cycling is well-established, the specific mechanisms governing microbial responses to freeze-thaw processes and their consequences for carbon sequestration remain largely unknown to date. Here, we highlight the importance of freeze-thaw processes for microbial community properties and carbon storage. These findings show important associations between microbial communities and active carbon storage, offering valuable insights for predicting how altered freeze-thaw patterns under climate warming might impact permafrost carbon storage.