SET and MYND domain-containing protein 3 (SMYD3) plays a key role in the progression of human cancer. Matrix metalloproteinase (MMP)-9 is being related to tumor progression. It has been reported that SMYD3 and MMP-9 are overexpressed in human cancers. However, the exact roles of SMYD3 and MMP-9 in the metastasis and prognosis of gastric cancer (GC) remain unclear. The expressions of SMYD3 and MMP-9 were detected by semiquantitative reverse transcription polymerase chain reaction and Western blotting in gastric cancer and adjacent nontumor tissues. In addition, SMYD3 and MMP-9 expressions were analyzed by immunohistochemistry in formalin-fixed samples from 186 gastric cancer patients. The messenger RNA (mRNA) and protein expression levels of SMYD3 and MMP-9 in gastric cancer tissues were both significantly higher than those in adjacent nontumor tissues. In addition, the expression of SMYD3 was correlated with size of primary tumor and lymph node metastasis, while size of primary tumor and serosal invasion were identified as the independently relative factors of MMP-9 expression in GC tissues. SMYD3 expression and MMP-9 expression in GC tissues were significantly and positively correlated. Multivariate analysis results demonstrated that degree of differentiation, lymph node metastasis, TNM stage, SMYD3 expression, and MMP-9 expression were the independent prognostic indicators of gastric cancer. SMYD3 and MMP-9 may play important roles in tumor invasion, metastasis, and prognosis and could work as promising targets for prognostic prediction in gastric cancer.