Regulatory T cells (Tregs) impair cancer immunosurveillance by creating an immunosuppressive environment fostering tumor cell survival. Our previous finding demonstrated that neutrophil extracellular traps (NETs), which are involved both in innate and adaptive immunity, are abundant in the NASH liver. However, how NETs can interact with Tregs in the development of nonalcoholic steatohepatitis-associated hepatocellular carcinoma (NASH-HCC) is not known.Stelic Animal Model, choline-deficient, high-fat diet + diethylnitrosamine mouse model, and western diet mouse model were utilized for NASH-HCC or NASH development. Treg depletion was achieved using FoxP3-DTR mice. RNA sequencing was used to explore the mechanism of NETs on Treg differentiation. Bioenergetic analyses of naïve CD4+ T cells were assessed by Seahorse.Although the absolute number of CD4+ T cells is decreased in NASH livers, the Treg subpopulation is selectively increased. Depleting Tregs dramatically inhibits HCC initiation and progression in NASH. There is a positive correlation between increased NET and hepatic Treg levels. RNA-seq data reveals that NETs impact gene profiles in naive CD4+ T cells and tilt the balance toward Tregs with most differential differentially expressed genes enriched in mitochondrial oxidative phosphorylation (OXPHOS). NETs through facilitating mitochondrial respiration can promote Treg differentiation. The metabolic reprogramming in naïve CD4+ T cells by NETs requires toll-like receptor 4 (TLR4). The blockade of NETs in vivo using PAD4-/- mice or DNase I treatment reduces the activity of Tregs.Tregs can suppress immunosurveillance in the premalignant stages of NASH. NETs facilitate the crosstalk between innate and adaptive immunity in NASH by promoting Treg activity through metabolic reprogramming. Therapies targeting NETs and Treg interactions can offer a potential strategy for preventing HCC in NASH.Regulatory T cells (Tregs) can promote tumor development by suppressing cancer immunosurveillance, but their role in carcinogenesis during NASH progression is unknown. Herein, we discovered that selectively increased intrahepatic Tregs can promote an immunosuppressive environment in NASH livers. Neutrophil extracellular traps (NETs) link innate and adaptive immunity by promoting Treg differentiation via metabolic reprogramming of naïve CD4+ T cells, which exposes a potential therapeutic intervention to prevent HCC in NASH.Copyright © 2021. Published by Elsevier B.V.