In 2019, the national cancer report showed that the incidence rate of gastric cancer (GC) ranked second, only after lung cancer. Its mortality rate ranked third in all tumors. It seriously endangers people’s health. It is very important for reducing the mortality rate of GC to screen and identify early detection markers and search for molecular targets for its treatment. CD90 (THY1) is a cell surface glycoprotein, which plays an important role in tumor cell proliferation, metastasis and angiogenesis. The abnormal expression of CD90 is related to the stem characteristics of cells and promotes the initiation and metastasis of tumor. However, the relationship between CD90 and the stem characteristics of GC cells has not been reported. In this study, we found that CD90 overexpression increased the proportion of side population cells (SP cells) of GC cells AGS. It affected the protein expression levels of stem markers such as ABCG2 and CD105 on the membrane surface, and also affected the mRNA expression levels of stem markers such as NANOG, and SOX2 in GC cells AGS. mRNA microarray and Western blot revealed that CD90 overexpression affected the stem characteristics of GC cells through activating the signal pathway of PI3K/Akt and JNK/ERK1. Our results provide a new idea for the identification of potential molecular targets of gastric cancer.