Objective To investigate the role of miR-374-5p in regulating mesenchymal stem cells ( MSCs) derived from gastric tissues to promote the proliferation and migration of gastric cancer cells in vitro. Methods The gastric cancer-mesenchymal stem cells ( GC-MSC) and the matched paracancerous tissue of gastric cancer derived normal mesenchymal stem cells ( GCN-MSC) were isolated and cultured in vitro. Gastric cancer cells were treated with the culture supernatants of GCN-MSC and GC-MSC respectively in vitro. The proliferation ability of gastric cancer cells was detected by plate clone test and the migration ability was detected by scratch test. MiR-374-5p mimics were used to over-express miR-374-5p in GC-MSC and GCN-MSC and MiR-374-5p inhibitor was used to inhibit its expression level. The culture supernatant of MSCs after transfection was collected and gastric cancer cells were treated in vitro. The changes of proliferation and migration ability of gastric cancer cells were observed. Results The number of single cell colonies formed in GC-MSC culture supernatant group (112±7) was more than that of GCN-MSC group (48±6) and the distance between the cells (6.5±0.5 cm) was less than that of GCN-MSC group (15±1) cm, by which the proliferation and migration of gastric cancer cells were significantly promoted (t=12.02, P<0.01 and t=13.17, P<0.01,respectively). Following miR-374-5p overexpressing in the culture supernatant of GCN-MSC,the number of single cell colonies increased (115±12.1) and space between cells reduced (7.83±1.08 cm), by which the proliferation ( t=9.31, P<0.01) and migration ( t=4.88, P<0.01) of gastric cancer were also promoted. The miR-374-5p inhibitor reversed the GC-MSC-induced ability of proliferation and migration of gastric cancer ( t=5.47,P<0.01 and t=4.95,P<0.01) . In the inhibitor group the number of single cell colonies decreased (60±10.2) and the space between cells increased (12.17±1.47cm) following treating by the culture supernatant. Conclusion MiR-374-5p should be an important molecule that regulates MSC in tumoral microenvironment to promote the proliferation and migration of gastric cancer and may become a prospective target for diagnosis and treatment of gastric cancer.