Gastric cancer is one of the world common causes of cancer death. Many people have suffered, but yet therapeutic methods found. May studies have showedthat Tanshinone IIA, a diterpenequinone, which extracted from the traditional herbal medicine Danshen (Salvia miltiorrhiza),has potential against many kind of cancer types. Our previous studies have demonstrated TIIA can kill gastric cancer AGS cells, but the response signalling is still unclear. Therefore, we used the time-dependent phosphoproteomic approach to reveal the regulatory effects of TIIA in AGS cells. Our results showed that a total of 1092 phosphoprotiens and 3332 phosphopeptides were identified in 3615 phosphorylation sites and 349 phosphosites corresponding to 220 phosphorylated proteins were significantly regulated. Furthermore, by using networkand functionalenrichmentanalyses, we found that TIIA regulated several cellular processesingastric cancer cells, such astranscription mRNA processing, DNA damage and protein unfolding response. Finally, we further validated that TIIA caused protein unfolding response and DNA damage via inducing ROS production. These findings not only uncover the molecular mechanisms mediated by TIIA but also contribute to the development of gastric cancer therapy.