Gastric cancer (GC) is the fourth most common cancer worldwide and is associated with a low 5-year survival rate of <24 % due to the tendency of early invasion and metastasis. Rab GTPases, which are master regulators of intracellular trafficking, have been shown to a play new role in the control of multiple tumor-related processes, including cell migration, invasion, proliferation, communication, and drug resistance. Here, we analyzed the mRNA expression levels of 63 Rab GTPases in samples from GC patients. Our data demonstrated that the expression level of Rab40b was significantly correlated with GC invasion classification (P < 0.01), lymph node metastasis (P < 0.01), and pathological stage (P < 0.01). High Rab40b mRNA expression was also correlated with shorter overall survival in patients with GC (P < 0.05). Moreover, knockdown of Rab40b protein reduced the migration and invasion of GC cells, while overexpression of Rab40b significantly promoted GC cell metastasis in nude mice. Our results also showed that Rab40b is a target gene of miR-204 and further demonstrated that Rab40b is negatively correlated with miR-204 in GC tissues. These findings indicate that Rab40b might be a novel prognostic marker and a candidate biological therapeutic target for GC.