Several liver diseases have been associated with the Tudor staphylococcal nuclease (Tudor‐SN) protein. Our previous results demonstrated that, in comparison to wild‐type (WT) mice, systemic overexpression of Tudor‐SN in transgenic (Tg) mice (Tudor‐SN‐Tg) ameliorates obesity‐induced insulin resistance and hepatic steatosis. In this study, we observed an inverse correlation in the expression levels of Tudor‐SN and profibrogenic factors, such as alpha‐smooth muscle actin (α‐SMA) and collagen alpha‐1(I) chain (COL1A1), in liver tissue samples between Tudor‐SN‐Tg and WT mice. The correlation was further validated in hepatic fibrotic tissues from patients with cirrhosis and fibrosis. Utilizing a carbon tetrachloride (CCl 4 )‐induced hepatic fibrosis model, we observed that Tudor‐SN attenuated hepatic fibrosis in mice. Tudor‐SN was abundantly expressed in hepatic stellate cells (HSCs). In the Tudor‐SN‐Tg group, primary HSCs showed stellate‐like morphology as well as reduced in vitro proliferation and chemotactic ability compared to the WT group. Pseudotime series analysis of HSCs further showed the role of Tudor‐SN during the dynamic evolution of HSC activation. Reduced Tudor‐SN expression facilitated the in vitro activation of LX‐2 cells. Furthermore, primary HSC cells from WT and Tudor‐SN knockout (KO) mice were isolated for RNA‐sequencing analysis. The findings suggested that Tudor‐SN may regulate the activation of primary HSCs by influencing lipid metabolism, translation initiation, immune response, and the extracellular matrix. In summary, we identified Tudor‐SN as a newly identified regulator involved in the transition of quiescent HSCs to activated states, shedding light on the antifibrotic impact of Tudor‐SN expression in the development of hepatic fibrosis.