BACKGROUND Gastric cancer (GC) has a poor prognosis and limited therapeutic options. As a new promising cancer therapeutic approach, chimeric antigen receptor (CAR)-T cells represent a potential GC treatment. We investigated the antitumor activity of CAR-T cells target-B7H3 in GC. METHODS In our study, expression of B7H3 was examined in GC tissues and explored the tumoricidal potential of B7H3-targeting CAR-T cells in GC. B7H3-directed CAR-T cells with a humanized antigen-recognizing domain was generated. The anti-tumor effects of this CAR-T cell were finally investigated in vitro and in vivo. RESULTS Our results show that B7H3-directed CAR-T cells efficiently killed GC tumor cells. In addition, we found that B7H3 is correlated with tumor cell stemness, and anti-B7H3 CAR-T can simultaneously target stem cell-like GC cells to improve the treatment outcome. CONCLUSIONS Our study indicates that B7H3 is an attractive target for GC therapy, and B7H3 has high potential for clinical application.