Data from the present study showed that agrimonolide exhibited a high anti-proliferation effect against human gastric cancer AGS cells. Flow cytometric analysis revealed that the number of total apoptotic cells increased after the treatment with the agrimonolide in a dose-dependent manner. In addition, it was found that agrimonolide-induced cell apoptosis was associated with the increase in the (Bcl-2 Associated X Protein, BAX)/(B-cell lymphoma-2, Bcl-2) ratio and the activation of cleaved caspase-3. MAPK (p38, c-Jun N-terminal kinase, and ERK1/2) signaling pathways were involved in agrimonolide-induced apoptosis. Cells were exposed to 40 μM of agrimonolide and the level of phospho-ERK/ERK protein was increased to 7.0-fold as compared to the control, and the expression of phospho-p38 protein showed a significant 6.2-fold increase after 24 h incubation, as compared to the control. The employment of protein kinase inhibitors of PD98059 and SB203580, showed the block effects of agrimonolide on the activation of caspase-3 and apoptosis.