BACKGROUND: Cryoablation plays a key role in the comprehensive management of lung adenocarcinoma, characterized by its ability to activate antitumor immunity. This study aimed to explore the impact of cryoablation on the local immune microenvironment, focusing on regulatory T cells (Tregs) and the TGF-β pathway. METHODS: Single-cell sequencing was employed to identify differences in immune cell populations and related pathway expression between lung adenocarcinoma tissues and adjacent noncancerous tissues. Prospective observations of changes in Tregs in the peripheral blood pre- and post-cryoablation for lung adenocarcinoma were conducted at Dongfang Hospital, Beijing University of Chinese Medicine. Bulk RNA-seq analysis of mouse tumor tissues was performed to predict the potential mechanisms underlying cryoablation-induced antitumor immunity. Finally, these predictions were validated through in vitro and in vivo experiments employing cell cryoablation and mouse subcutaneous tumor transplantation models. RESULTS: Single-cell RNA sequencing analysis revealed intricate interactions between Tregs subpopulations and the regulation of the immune response in lung adenocarcinoma, highlighting the involvement of the TGF-β pathway. A significant decrease in the level of Tregs was noted at 30 days post-cryoablation compared to pre-surgical and 3-day post-surgery levels. The cellular and murine cryoablation models validated the inhibitory effect of cryoablation on Tregs and its potential to stimulate antitumor immunity. Additionally, the results of bulk RNA-seq demonstrated the role of cryoablation in regulating postoperative immunity via the TGF-β pathway. Cryoablation decreased the expression levels of TGF-β1, suppressed the phosphorylation of Smad2 and Smad3, and downregulated the expression of FOXP3, thereby inhibiting the conversion of CD4 + T cell precursors into Tregs. Moreover, cryoablation enhanced the expression of interferon-gamma (IFN-γ), thereby promoting its antitumor activity. CONCLUSIONS: This study revealed the effective modification of the lung adenocarcinoma microenvironment by cryoablation through the suppression of Tregs and activation of antitumor immunity via the TGF-β pathway. These findings hold implications for optimizing cryoablation-based therapies and guiding future clinical trials on lung adenocarcinoma treatment. TRIAL REGISTRATION: This trial was registered with the Chinese Clinical Trial Registry (Chictr.org.cn, ChiCTR2000038580, Sep 24, 2020).