To establish an RP-HPLC method for the determination of β-sitosterol content in Fructus Bruceae extract; and to observe its inhibitory effect on gastric cancer metastasis. β-sitosterol content in Fructus Bruceae extract was determined by RP-HPLC. Inhibitory effects of low-, medium- and high-dose extracts on the adhesion of human gastric cancer SGC-7910 cells to Fibronectin (FN) were detected by MTT assay. Inhibitory effects of the extract on SGC-7910 cell invasion and migration were observed by reconstituted basement membrane invasion assay. Absorbance values of low-, medium- and high-dose Fructus Bruceae extract treatment groups were significantly different compared to the control group (P<0.01). After treatment with low-, medium- and high-dose extracts, the number of cells passing through the membrane differed significantly from the control group (P<0.05). After treatment with high-dose extract, the scratch area spacing was significantly different from the control group (P<0.01). Fructus Bruceae extract can inhibit the adhesion, invasion and migration of gastric cancer cells, which has an inhibitory effect on the gastric cancer metastasis. The RP-HPLC method established provides a reliable basis for the quantitative evaluation of Fructus Bruceae.