Mesenchymal stem cells (MSCs), with the capacity for self-renewal and differentiation into multiple cell types, exhibit the property of homing towards tumor sites and immunosuppression and have been used as tumor-tropic vectors for tumor therapy. However, few studies have investigated the underlying molecular mechanisms that link MSCs to targeted tumor cells. In this study, we elucidated the inhibitory effects and mechanisms of human bone marrow mesenchymal stem cells (hBMSCs) on human glioma U251 cells using a co-culture system in vitro. The anti-tumor activity of co-cultured hBMSCs was assessed by morphological changes, the MTT assay, and Hoechst 33258 staining. Cell apoptosis and cell cycle distribution were evaluated by flow cytometry. Cell migration and invasion were evaluated using a 24-well Transwell chamber. A proteomics approach was used to identify differentially expressed proteins after hBMSCs treatment in U251 cells, and quantitative polymerase chain reaction was used to validate the results. Bioinformatics analyses were also implemented to better understand the identified proteins, and Western blotting analyses were used to analyze the associated proteins. The results showed that hBMSCs could inhibit cell proliferation and induce cell cycle arrest in the G1 phase, resulting in apoptosis of U251 cells. Transwell and Matrigel invasion assays showed that hBMSCs reduced the migration and invasion of U251 cells. Using proteomics, 11 differentially expressed proteins were identified and observed. Bioinformatics analyses indicated that the identified proteins participated in several biological processes and exhibited various molecular functions, mainly related to the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway. Moreover, hBMSCs regulated changes in proteins linked to cell apoptosis and cell cycle progression and inhibited the epithelial-mesenchymal transition (EMT)-like and PI3K/AKT pathway. Taken together, the findings in our study suggest that hBMSCs inhibit U251 cells proliferation and the EMT-like by downregulating the PI3K/AKT signaling pathway, which indicates that hBMSCs have a potential antitumor characteristics and should be further explored in future glioma therapy.