Apart from other risk factors, mechanical stress on joints can promote the development of osteoarthritis (OA), which can also affect the temporomandibular joint (TMJ), resulting in cartilage degeneration and synovitis. Synovial fibroblasts (SF) play an important role in upkeeping joint homeostasis and OA pathogenesis, but mechanical stress as a risk factor might act differently depending on the type of joint. We thus investigated the relative impact of mechanical stress on the gene expression pattern of SF from TMJs and knee joints to provide new insights into OA pathogenesis. Primary SF isolated from TMJs and knee joints of mice were exposed to mechanical strain of varying magnitudes. Thereafter, the expression of marker genes of the extracellular matrix (ECM), inflammation and bone remodelling were analysed by quantitative real-time polymerase chain reaction (RT-qPCR). SF from the knee joints showed increased expression of genes associated with ECM remodelling, inflammation and bone remodelling after mechanical loading, whereas TMJ-derived SF showed reduced expression of genes associated with inflammation and bone remodelling. SF from the TMJ differed from knee-derived SF with regard to expression of ECM, inflammatory and osteoclastogenesis-promoting marker genes during mechanical strain. Osteoarthritis-related ECM remodelling markers experience almost no changes in strain-induced gene expression, whereas inflammation and bone remodelling processes seem to differ depending on synovial fibroblast origin. Our data indicate that risk factors for the development and progression of osteoarthritis such as mechanical overuse have a different pathological impact in the TMJ compared to the knee joint.