We studied centrifugation-mediated mechanical stress induced Tumor necrosis factor (TNF)- production in the monocyte-like cell line THP-1. The induction of TNF- by mechanical stress was dependent on the centrifugation speed and produced the highest level of TNF- after 1 h of stimulation. TNF- production returned to normal levels after 24 h of stimulation. Mechanical stress also induced Toll-like Receptor (TLR) 2 mRNA in proportion to the expression of TNF-. The inhibition of TLR2 signaling by dominant negative myeloid differentiation factor 88 (MyD88) blocked TNF- expression response to mechanical stress. After transient over-expression of TLR2 in HEK293 cells, mechanical stress induced TNF- mRNA production. Interestingly, mechanical stress activated the c-Src-dependent TLR2 phosphorylation, which is necessary to induce Ca²⁺ fluxes. When THP-1 cells were pretreated with BAPTA-AM, thapsigargin, and NiCl2•6H2O followed by mechanical stimulation, both TLR2 and TNF- production were inhibited, indicating that centrifugation-mediated mechanical stress induces both TLR2 and TNF- production through Ca²⁺ releases from intracellular Ca²⁺ stores following TLR2 phosphorylation. In addition, TNF- treatment in THP-1 cells induced TLR2 production in response to mechanical stress, whereas the pre-incubation of anti-TNF- antibody scarcely induced the mechanical stress-mediated production of TLR2, indicating that TNF- produced by mechanically simtulated-THP-1 cells affected TLR2 production. We concluded that TNF- production induced by centrifugation-mediated mechanical stress is dependent on MyD88-dependent TLR2 signaling that is associated with Ca²⁺ release and that TNF- production induced by mechanical stress affects TLR2 production.