Exploring cellular senescence inducers is one of the approaches to screen anti-tumor active lead compounds. Whether glaucocalyxin B induces cellular senescence remains to be elucidated. This study aims to investigate the effect and mechanism of glaucocalyxin B on the senescence of lung adenocarcinoma A549 cells. 2 and 5 μmol·L^-1 of glaucocalyxin B induced senescence in A549 cells. Glaucocalyxin B significantly increased the levels of cyclin-dependent kinase inhibitor 1A (p21) and the proportion of β-galactosidase staining-positive cells in A549 cells. Glaucocalyxin B had no significant effect on the levels of peroxiredoxin Ⅰ (Prdx Ⅰ) protein in A549 cells but significantly inhibited its enzymatic activity. Knockdown of Prdx Ⅰ induced senescence in A549 cells. Glaucocalyxin B inhibited the enzyme activity of Prdx Ⅰ, leading to the increase of reactive oxygen species (ROS), and the ROS scavenger N-acetylcysteine attenuated glaucocalyxin B-induced the senescence of A549 cells. Either glaucocalyxin B or Prdx Ⅰ knockdown significantly upregulated the levels of tumor protein p53 in A549 cells, while knockdown of p53 significantly inhibited glaucocalyxin B-induced the senescence of A549 cells. In conclusion, glaucocalyxin B can induce senescence in lung adenocarcinoma cells by inhibiting the Prdx Ⅰ/ROS/p53 pathway, which might be one of its anti-tumor mechanisms.