Esophageal-gastric junction adenocarcinoma (AEG) is an aggressive tumor with high incidence and dismal prognosis worldwide. Despite significant advances in therapeutic strategies, the 5-year survival rate still remains low. Diallyl disulfide (DADS), which is one of the major volatile components isolated from garlic, has been shown to have multi-targeted antitumor activities in a variety of cancer cells. However, the exact anti-metastatic effects and underlying molecular mechanisms of DADS in AEG have not been elucidated. The present study demonstrated that DADS inhibited cell viability of OE19 cells with low cytotoxicity to healthy hepatocytes, L02 cells, in vitro. Non-toxic doses of DADS were ≤10 µg/ml for a 24-h treatment. Our data showed that these non-toxic doses of DADS were found to block the metastasis of OE19 cells by suppressing MMPs, increasing u-PA and TIMPs, as well as altering the balance of MMPs/TIMPs, which at least in part resulted from the suppression of NF-κB and PI3K/AKT signaling pathways. The present study provides a previously neglected insight into the investigation of DADS in suppressing tumor metastasis and its underlying molecular mechanisms in vitro. Hence, DADS could be a promising anticancer agent for anti-metastatic treatment of AEG in the future.