Atractylenolide II (AT-II) exhibits several biological and pharmacological functions, especially anti-cancer activity as the major sesquiterpene lactones isolated from Atractylodes macrocephala (also named Baizhu in Chinese). However, the effects and mechanisms of AT-II on human gastric cancer remain unclear. Cell Counting Kit-8 (CCK-8) assay, morphological changes, flow cytometry, wound healing assay and Western blot analysis were used to investigate the effects of AT-II on cell proliferation, apoptosis and motility of human gastric carcinoma cell lines HGC-27 and AGS. Our results indicated that AT-II could significantly inhibit cell proliferation, motility and induce apoptosis in a dose and time-dependent manner. Western blot analysis showed that the expression level of Bax was upregulated and the expression levels of B-cell lymphoma-2 (Bcl-2), phosphorylated-protein kinase B (p-Akt) and phosphorylated-ERK (p-ERK) were downregulated compared to control group. In conclusion, the findings suggested that AT-II exerted significant anti-tumor effects on gastric carcinoma cells by modulating Akt/ERK signaling pathway, which might shed light on therapy of gastric carcinoma.