Epidemiological studies demonstrate that the incidence and mortality of gastric cancer in women are lower than in men worldwide. Many studies have reported the delayed menopause and hormone replacement therapy are associated with a reduced risk for gastric cancer. It has been reported that endogenous estrogen lowers gastric cancer incidence in women, and cancer patients treated with estrogens have a lower subsequent risk of gastric cancer. It has been reported that estrogen decreases the progression of gastric cancer by inhibiting erbB-2 oncogene expression. Overexpression of estrogen receptor might inhibit the proliferation and invasion of MKN28 gastric cancer cells. Accumulating evidence suggests that bone marrow mesenchymal stem cells contribute to the progression of gastric cancer. However, it is unknown if 17β-estradiol (E2) treatment is sufficient to inhibit human bone marrow mesenchymal stem cells (HBMMSCs)-mediated cell motility in human gastric cancer cells. The results from human cytokine arrays have shown that HBMMSCs notably secrete interleukin 6 (IL-6) protein. Administration of IL-6–specific neutralizing antibody significantly inhibits HBMMSCs-mediated motility activity in human gastric cancer cells. Treatment of recombinant IL-6 soluble protein confirmed the role of IL-6 in mediating HBMMSCs-upregulated cell motility. IL-6 mainly upregulates motility activity via activation of Src signaling pathway in human gastric cancer cells. We further observed that E2 treatment inhibits HBMMSCs-induced cellular motility via suppressing the activation of IL-6−Src/Cas/paxillin signaling pathway in human gastric cancer cells. Collectively, these results suggest that E2 treatment significantly inhibits HBMMSCs-induced cellular motility in human gastric cancer cells.