Tumor necrosis factor alpha (TNFα) is a pro-inflammatory cytokine responsible for immune regulation and is considered to execute its function mainly through its receptor- mediated canonical signal pathway. Mesenchymal stem cells (MSCs) are the heterogeneous primitive cells initially discovered residing in the adult bone marrow stroma, possessing self-renewal and multiple differentiation potential and critically maintaining multiple tissue/organ homeostasis. The interplay between MSCs and immune cytokines via the receptors on the MSC surface has increasingly been recognized; increasing evidence has shown that MSCs produce a certain amount of cytokines by themselves with little understanding of the role of MSC-derived cytokines. In this study, we, for the first time, reveal a non-inflammatory, non-canonical role of MSC-derived TNFα by showing that TNFα-deficient MSCs exhibit impaired self-renewal and differentiation due to upregulated mTOR phosphorylation. Mature TNFα is internalized into the cytoplasm via endocytosis after being cleaved by the TNFα- converting enzyme and shedded into the extracellular microenvironment. We further find that cytoplasmic TNFα binds to Rictor, a component of mTOR complex 2, to restrain mTOR activation. A complex regulatory network and signaling pathways are involved in governing MSC fate commitment. Mechanical stimuli, including physical cues from the matrix and applied forces, account for one critical extrinsic factor controlling MSC fate determinations. Microgravity conditions, such as astronauts in spaceflight missions and bedridden patients, are reported to result in progressive bone loss, but the therapeutics have yet to be established. In our study, we use hindlimb unloading (HU) mice to mimic the microgravity condition and find that HU mice resulted in reduced TNFα endocytosis and impaired cell function in MSCs as well as osteopenia phenotype. Rapamycin therapy rescues MSCs impairment and osteopenia in HU mice by blocking mTOR activation. Collectively, our findings identify a previously unrecognized role of TNFα in maintaining MSC homeostasis via receptor-independent endocytosis to finetune mTOR signaling homeostasis. A mechanical stimuli-dependent and receptor-independent endocytosis of TNFα is required to maintain mTOR equilibrium and therefore safeguard MSC homeostasis. Rapamycin may be a promising therapy for hypodynamia-induced osteoporosis in astronauts and bedridden patients.