Background: The purpose of this study was to evaluate the clinical significance and prognostic roles of vasculogenic mimicry (VM) and elucidate their intrinsic association with molecular markers. Methods: 89 human gastric cancer cases with detailed follow-up and clinicopathologic data were collected. CD34/periodic acid-Schiff double staining was performed to validate the existence of VM. Immunohistochemistry was performed to explore the expression of different molecular factors. Results: VM was observed in 24 gastric cancer patients. They were prone to higher histological grade, hematogenous metastasis, distant recurrence, and chance of progression to stage III or IV (p < 0.05). The VM group had shorter overall and disease-free survival (p < 0.05). VM negativity was independently prognostic for prolonged overall or disease-free survival (p < 0.05). VM was positively associated with levels of matrix metalloproteinase-2, matrix metalloproteinase-9, vascular endothelial growth factor, and vascular endothelial growth factor receptor-1 (p < 0.05), but not with vascular endothelial growth factor receptor-2 (p > 0.05). Conclusion: VM should be regarded as a good marker to indicate pathobiological behaviors of gastric cancer. Using antibodies against matrix metalloproteinases, vascular endothelial growth factor, or vascular endothelial growth factor receptors could be strategies to counteract VM formation.