Oxymatrine (OMT), is a natural quinoxaline alkaloid from the traditional Chinese medicine herb and has been shown to exhibit anticancer properties on various types of cancer cells. Poorly differentiated gastric adenocarcinoma is a common malignancy of gastric cancer that is more aggressive and has a poor prognosis. In the present study, we investigate the effects of Slit2/Robo1 signals in poorly differentiated gastric adenocarcinoma and adjacent tissues, and the anticancer properties of OMT on human poorly differentiated gastric adenocarcinoma BGC-823 cells and evaluate their underlying mechanisms. The expression levels of Slit2 and Robo1 proteins were measured in 20 pairs of human poorly differentiated gastric adenocarcinoma tissues and adjacent normal tissues using western blot. The expression of apoptosis related proteins and autophagy-related proteins was detected by western blot. The cells viability was detected by CCK-8 assay. The migration of BGC-823 cells was detected by transwell experiments. The expression of related proteins was detected by western blot. The result shows that Slit2 and Robo1 are significantly increased in poorly differentiated gastric adenocarcinoma. The apoptosis and autophagy are inhibited in poorly differentiated gastric adenocarcinoma. OMT inhibits the growth and migration of BGC-823 cells in vitro. OMT inhibits the activation of Slit2/Robo1 signals and induces apoptosis and autophagy in BGC-823 cells. These findings suggest that the antitumor effects of OMT may be the result of inhibition of cell growth and migration, and inhibits the activation of Slit2/Robo1 signals pathway and induces apoptosis and autophagy. OMT may represent a novel anticancer therapy for the treatment of poorly differentiated gastric adenocarcinoma.