Dysregulated expression of microRNAs (miRNAs) has been shown to be closely associated with tumor development, progression, and carcinogenesis. However, their clinical implications for gastric cancer remain elusive. To investigate the hypothesis that genome-wide alternations of miRNAs differentiate gastric cancer tissues from those matched adjacent non-tumor tissues (ANTTs), miRNA arrays were employed to examine miRNA expression profiles for the 5-pair discovery stage, and the quantitative real-time polymerase chain reaction (qRT- PCR) was applied to validate candidate miRNAs for 48-pair validation stage. Furthermore, the relationship between altered miRNA and clinicopathological features and prognosis of gastric cancer was explored. Among a total of 1,146 miRNAs analyzed, 16 miRNAs were found to be significantly different expressed in tissues from gastric cancer compared to ANTTs (p<0.05). qRT-PCR further confirmed the variation in expression of miR-193b and miR-196a in the validation stage. Down-expression of miR-193b was significantly correlated with Lauren type, differentiation, UICC stage, invasion, and metastasis of gastric cancer (p<0.05), while over-expression of miR-196a was significantly associated with poor differentiation (p=0.022). Moreover, binary logistic regression analysis demonstrated that the UICC stage was a significant risk factor for down-expression of miR-193b (adjusted OR=8.69; 95%CI=1.06-56.91; p=0.043). Additionally, Kaplan-Meier survival curves indicated that patients with a high fold-change of down-regulated miR-193b had a significantly shorter survival time (n=19; median survival=29 months) compared to patients with a low fold-change of down-regulated miR-193b (n=29; median survival=54 months) (p=0.001). Overall survival time of patients with a low fold-change of up-regulated miR- 196a (n=27; median survival=52 months) was significantly longer than that of patients with a high fold-change of up-regulated miR-196a (n=21; median survival=46 months) (p=0.003). Hence, miR-193b and miR-196a may be applied as novel and promising prognostic markers in gastric cancer.