Gastric cancer (GC) is one of the most aggressive tumors and has a poor prognosis. It has been demonstrated that gastric cancer mesenchymal stem cells (GC-MSCs) can promote the progression, metastasis, and chemoresistance of GC through various mechanisms, but the effect of GC-MSCs on GC during chemotherapy is still unknown. In this study, flow cytometry, CCK8 assay, migration assay, colony formation assay, and western blot were conducted. We also analyzed GC patients from the cancer genome atlas (TCGA). Our results showed that GC-MSCs were resistant to 5-FU and Taxol at the IC50 concentration for GC cells, and 5-FU could promote the migration of GC-MSCs at low doses. Furthermore, the conditioned medium of GC-MSCs pretreated with chemotherapeutic drugs was more effective in promoting the proliferation, migration, and stemness of GC cell lines than the conditioned medium of GC-MSCs without chemotherapeutic drugs treatment. These effects were dependent on the activation of phosphorylated AKT (p-AKT) in GC cell lines. Correspondingly, the inhibition of p-AKT reversed the tumor-promoting effect of the conditioned medium of GC-MSCs pretreated with chemotherapeutic drugs. Additionally, the expression of AKT1 was higher in GC tissues than in both paracancerous tissues and normal tissues, and patients resistant to chemotherapy expressed more AKT1 compared to those who were sensitive. Taken together, our data demonstrated that GC-MSCs gained more tumor-promoting abilities during chemotherapy.