α-fetoprotein (AFP) is a valuable tumor marker for many types of cancers, including primary gastric cancer (GC). However, the effects of AFP expression on the metastasis and anoikis sensitivity of GC remain unclear. The present study aimed to explore the role and possible mechanism of AFP in the invasion and metastasis of GC AGS cells, particularly in the anoikis sensitivity of AGS cells. In the present study, the expression of AFP in cultured AGS cells was assayed firstly by RT-PCR, western blotting and sequencing. Then, a specific AFP siRNA was applied to interfere with AFP expression and poly(2-hydroxyethyl methacrylate) (poly-HEMA) was used to block cell anchorage. The invasion and metastatic ability, and anoikis sensitivity detections were conducted based on Transwell chamber assay, anoikis assay kit and western blotting. Our results confirmed the expression of AFP in AGS cells. Then, we found that interference of AFP with siRNA attenuated the invasion and metastasis of AGS cells and induced a significant upregulation of E-cadherin and downregulation of N-cadherin expression (P<0.05). Cell apoptosis and anoikis were induced when cell anchorage was blocked by poly-HEMA treatment, which was exacerbated significantly when cells were exposed to AFP siRNA. Moreover, interference of AFP when cell anchorage was blocked enhanced the expression of the pro-apoptotic proteins Bax, caspase-3 and -9, and decreased the expression of the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2). In conclusion, the present study demonstrated that interference of AFP reduced AGS cell invasion and metastasis by enhancing anoikis sensitivity. The present study provides new insight for the treatment of GC and suggests AFP as a potential therapeutic target by regulating anoikis sensitivity.