Gastric cancer is a common malignancy with a poor prognosis. β-elemene is a broad-spectrum anticancer drug extracted from the traditional Chinese medicinal herb Curcuma wenyujin. In the present study, we investigated the anticancer effects of β-elemene in gastric cancer cells and the potential proteins involved. Human SGC7901 and MKN45 gastric cancer cells were treated with different concentrations of β-elemene. Cell viability, clonogenic survival and apoptotic cell death were assessed. β-elemene inhibited viability and decreased clonogenic survival of gastric cancer cells in a dose-dependent manner. Apoptosis induction contributed to the anticancer effects. We then employed a proteomic method, isobaric tags for relative and absolute quantitation (iTRAQ), to detect the proteins altered by β-elemene. In total, 147 upregulated proteins and 86 downregulated proteins were identified in response to β-elemene treatment in SGC7901 gastric cancer cells. Among them, expression of p21-activated protein kinase‑interacting protein 1 (PAK1IP1), Bcl-2-associated transcription factor 1 (BTF) and topoisomerase 2-α (TOPIIα) were validated by western blot analyses and the trends were consistent with iTRAQ results. Top pathways involved in β-elemene treatment in SGC7901 gastric cancer cells included ribosome signaling, peroxisome proliferator-activated receptors (PPARs) signaling pathway, regulation of actin cytoskeleton, phagosome, biosynthesis and metabolism of some amino acids. Collectively, our results suggest a promising therapeutic role of β-elemene in gastric cancer. The differentially expressed proteins provide further insight into the potential mechanisms involved in gastric cancer treatment using β-elemene.