Early responses to tendon overloading involve functional changes of tenocytes from anabolic (producing matrix protein) to catabolic (breaking down matrix proteins) state. These changes are thought to be induced by a combination of changes in extracellular mechanical environment as well as extrinsic inflammatory stimulation such as interleukin-1 β (IL-1 β), which stimulates tenocyte catabolism. However, detailed mechanisms are still largely unknown. We have focused on cellular tension as a possible regulator of tenocyte catabolism and inflammatory responses. The present study was performed to investigate if tenocyte response to IL-1 β stimulation can be regulated by cellular tension. Tenocytes isolated from rabbit Achilles tendons were seeded onto one of the following substrates: glass or PDMS-made micropillar substrate (3 µm diameter, 6 µm spacing in a hexagonal lattice with a pillar height of 2, 4 or 8 µm). Substrate stiffness was the highest in glass and the lowest in 8 µm-height micropillars. Following a 24-h incubation, IL-1β was administrated at 0 pM (control), 1 pM, 10 pM or 100 pM. IL-1β culture was performed for 3 days. Cell shapes and mRNA expression of matrix metalloproteinase-1 (MMP-1) in each condition was assessed. It was demonstrated that cell shape was remarkably influenced by both substrate stiffness and the concentration of IL-1 β. Cell area was significantly decreased with lowering substrate stiffness and increasing IL-1β concentration. The expression of MMP-1 mRNA was also influenced by both the substrate stiffness and ILl β concentration. These findings suggest that cellular tension, which is thought to reflect the substrate stiffness, is a key mechanical factor in the regulation of tenocyte functions, in particular their responses to inflammatory stimulation.