Pseudolaric acid B (PAB) is the major bioactive constituent in the root bark of Pseudolarix kaempferi and has been reported to have cytotoxicity against tumor cells. Our in vivo experiments showed that PAB could inhibit gastric cancer cell lung metastasis in a nude mouse haematogenous dissemination model. To evaluate the anti-metastasis mechanism of PAB in gastric cancer cells, cytological experiments were performed. The results showed that PAB could inhibit the adhesion ability to matrigel, migration, invasion and colony formation ability of BGC-823 and MKN-45 cells. Western blot further confirmed that the inhibitory effects of PAB on anti-metastasis may involve regulating the expression of the metastasis-related proteins MMP-9, HIF-1α, VEGF, VEGFR2, E-Cadherin and Ezrin. We obtained further proof that PAB which could be used as a multi-targeted agent to inhibit the PI3K/AKT, ERK1/2 and mitochondria-mediated apoptosis pathways and consequently suppress tumor growth and metastasis. Our experiments suggest that PAB-induced effects may have novel therapeutic applications for the treatment of gastric cancer.