Although there have been many reports about the relationship between force generation by skeletal muscles and aging, no study has investigated the relationship between contraction forces generated by non-muscle cells and aging. In this study, we examined that relationship using fibroblast populating collagen gels and a contraction force detecting system. Fibroblasts at passages 5 to 7 were used as the young group and those at passages 17 to 19 were used as the aged group. The contraction force induced by thrombin or lysophosphatidic acid significantly decreased with age. The expression of myosin light chain kinase (MLCK) and two types of Rho kinases (Rock-1 and Rock-2) decreased with age, but the expression of Rho A and myosin phosphatase (MPPase) did not change at all. The expression of myosin light chain 20k (MLC(20)) depended on the donor fibroblasts. Fibroblasts from young or aged hairless mice showed similar age-dependent results. Taken together, our data suggest that decreased expressions of MLCK and Rho kinase are critical for loss of force generation by fibroblasts with aging, which suggests new mechanisms of functional deficiencies due to aging.