The stiffness of the extracellular matrix can profoundly influence cell and tissue behaviors. Thus there is an emerging emphasis on understanding how matrix mechanical environments are established, regulated, and modified. Here we develop a microrheo‐metric assay to measure the mechanical properties of a model extracellular matrix (type I collagen gel) and use it to explore cytokine‐induced, cell‐mediated changes in matrix mechanical properties. The microrheometric assay uses micron‐scale ferrimagnetic beads embedded within collagen gels during fibrillogenesis. The beads are magnetized, then subjected to a twisting field, with the aggregate rotation of the beads measured by a magnetometer. The degree of bead rotation reflects the stiffness of the surrounding matrix. We show that the microscale assay provides stiffness measures for collagen gels comparable to those obtained with standard macroscale rheometry. To demonstrate the utility of the assay for biological discovery, we measure stiffness changes in fibroblast‐populated collagen gels exposed to three concentrations of six cytokines over 2 to 14 days. Among the cytokines tested, transforming growth factor‐β1 and interleukin‐1 β enhanced matrix stiffness, and together exerted cooperative effects on cellular modulation of matrix mechanics. The microrheometry approach developed here should accelerate the discovery of biological pathways orchestrating cellular modulation of matrix mechanics.–Leung, L. Y., Tian, D., Brangwynne, C. P., Weitz, D. A., Tschumperlin, D. J. A new microrheometric approach reveals individual and cooperative roles for TGF‐β1 and IL‐1β in fibroblast‐mediated stiffening of collagen gels. FASEB J. 21, 2064–2073 (2007)