Background and Aims Anoikis effector Bcl-2 transcription inhibitor 1 (Bit1) is abnormally expressed in gastric cancer tissue, the function and mechanism of Bit1 in gastric cancer is worth studying. This study was conducted to further examine the expression of Bit1 in gastric cancer tissue, and present a preliminary analysis of the effects of Bit1 on the biological behavior of gastric cancer cells.Methods The expressions of Bit1 in the surgical specimens of gastric cancer and corresponding adjacent tissue from 20 patients with primary gastric cancer were determined by Western blot analysis. In gastric cancer BGC-803 cells after transfection with Bit1 shRNA lentivirus or empty lentivirus vector, with the untreated wild type BGC-803 cells as blank control, the transfection efficiency and Bit1 silencing effect were validated by immunofluorescence assay, and then, the invasion, migration, proliferation and apoptosis were Transwell invasion assay, scratch wound assay, CCK-8 assay and TUNEL staining assay, respectively.Results The results of Western blot showed that the expression level of Bit1 protein in gastric cancer tissue was significantly up-regulated compared with corresponding adjacent tissues (P<0.01). The results of immunofluorescence assay demonstrated the successful transfection and effective Bit1 silencing. The results of series of cellular behavior experiments revealed that the wound-healing rate, number of invaded cells, and proliferation ability were all significantly decreased, while the number of late apoptotic cells was significantly increased in BGC-803 cells with Bit1 shRNA transfection compared with blank control cells (all P<0.05); there were no statistical differences in these cellular behavior parameters between BGC-803 cells transfected with empty vector and blank control cells (all P>0.05).Conclusion The expression level of Bit1 is up-regulated in gastric cancer, and it is closely related to the malignant biological behavior of gastric cancer cells, suggesting that Bit1 may be a biomarker associated with the initiation, progression and prognosis as well as a candidate therapeutic target for gastric cancer.