The aim of this study was to investigate the expression of nucleolin in tumorous tissues and corresponding non-malignant tissues in gastric cancer (GC), and the correlation of different expression sites with clinicopathologic parameters and prognosis. Immunohistochemistry was used for detecting the expression levels of nucleolin in GC tissues and corresponding non-malignant tissues from 124 gastrectomy specimens with stage I–III. Staining results were correlated with clinicopathologic features and survival. Both GC tissues and corresponding non-malignant tissues showed nucleolar staining for nucleolin. Nucleolin expression was higher in GC tissues than in non-malignant tissues. Among the 124 GCs, 85 (68.5%) were nucleolin-high. No significant correlation between nucleolin expression and other clinicopathologic parameters was found. The Cox univariate analysis indicated that both cytoplasmic staining and nucleolar staining of nucleolin expression correlated with patients' prognosis (log-rank, p < 0.0001; p = 0.0075, respectively). It was concluded in the study that nucleolin was overexpressed in GCs. A high level of nucleolar expression of nucleolin was an independent prognostic marker for better survival while high cytoplasmic staining was closely associated with worse prognosis for GC patients.