Complementary DNA microarrays have identified aberrantly expressed genes in patients with gastric cancer. One that encodes secretory leukocyte protease inhibitor (SLPI) is among the aberrantly expressed genes and is associated with metastasis in gastric cancers. We evaluated the potential of SLPI expression as a helpful biomarker for detection of gastric cancer. Tumor tissue and matching noncancerous mucosa were obtained from 60 patients immediately after gastric resection. SLPI expression levels were determined by Northern and Western blot tests and quantitative-reverse transcriptase-polymerase chain reaction (Q-RT-PCR). Paraffin-fixed tumor tissues were used for immunohistochemistry study in 119 patients. A consistent result was obtained between all examinations except plasma SLPI. SLPI mRNA transcripts and protein were overexpressed in gastric cancer cells, and the depth of wall invasion was significantly greater in serosa-invading (T3 and T4) cancers compared to the serosa-free (T1 and T2) cancers. These enhanced expressions were significantly associated with lymph node metastasis, and were significantly higher in stages III and IV, and higher than those in stages I and II. Five-year survival of patients with lower expression of the SLPI gene was significantly better than among patients with a higher expression. To better understand the function of SLPI in human gastric cancer cells, isogenic SLPI overexpressing cell lines (AZ521) were prepared. The migratory and invasive abilities were increased 4.4-fold to 6.9-fold, or 3.0-fold to 4.1-fold, respectively, in SLPI-overexpressing cell lines. The results point to SLPI as a potential prognostic marker for gastric cancer and its function in cell invasion.