The heat shock protein 90 (HSP90) is overexpressed and highly associated with poor prognosis in many malignancies. However, the role of HSP90 in gastric cancer has not been thoroughly elucidated. The aim of this study is to investigate the relationship of HSP90 expression with clinicopathological parameters and prognosis in advanced gastric cancer, and estimate the alteration of HSP90 expression after neoadjuvant chemotherapy. HSP90 and matrix metallopeptidase 9 (MMP-9) antigen expression was evaluated by immunohistochemistry in 322 advanced gastric carcinoma samples. The relationships between HSP90 and clinicopathological parameters and prognosis were analyzed. The response of HSP90 level was assessed in chemotherapeutic effect in 54 patients received 1-2 cycles of neoadjuvant chemotherapy. The positive expression of HSP90 was found to be 69.6% in 322 advanced gastric carcinoma samples. HSP90 protein expression was significantly associated with depth invasion (P<0.001), lymph node metastasis (P<0.001) and stage of disease (P<0.001). The positive rates of HSP90 expression were higher in both prominent serosal invasion group (P<0.001) and lymph node metastasis group (P<0.001). Moreover, HSP90 was significantly correlated with MMP-9 among 322 gastric cancer tissues (P<0.001). In univariate and multivariate analyses, HSP90 was an independent prognostic factor for both recurrence-free survival (RFS) and overall survival (OS). These results suggested that HSP90 may play an important role on tumor invasion, metastasis and prognosis, and might act as a promising target for prognostic prediction.