Gastric cancer is the fourth most common cancer worldwide. Several signaling pathways are involved in gastric cancer development and progression. Slit2 was recently found to be involved in cancer; however, its expression pattern in gastric cancer has not been discovered yet. In the present study, we investigated the expression of Slit2 in human gastric cancer and its correlation with the expression and subcellular localization of β-catenin. Immunohistochemistry (IHC) staining revealed that Slit2 was highly expressed in human gastric cancer tissues, while it was low or weakly expressed in normal gastric tissues. The differences in clinicopathological features between different groups were determined using Pearson's χ2 test. Slit2 levels were significantly associated with differentiation, Lauren's classification, lymph node metastasis and TNM staging. Slit2 levels were positively correlated with β-catenin level in gastric cancer tissues and cell lines. High levels of Slit2 were correlated with the membrane localization of β-catenin, and low levels of Slit2 were correlated with nuclear translocation of β-catenin in both gastric cancer tissues and cell lines assayed by IHC and immunofluorescence staining, respectively. Our data suggest that Slit2 was highly expressed in gastric cancer patients with less advanced clinicopathological features. Slit2 levels were correlated with β-catenin level and subcellular localization.