Podosomes are dynamic actin-based membrane protrusions that are important for extracellular matrix degradation and invasive cell motility. Individual podosomes are often found to organize into large rosette-like structures in some types of cells, such as osteoclasts, endothelial cells, Src-transformed fibroblasts, and certain highly invasive cancer cells. In this study, we show that new podosome rosettes arise through one of two mechanisms; de novo assembly or fission of a pre-existing podosome rosette in Src-transformed fibroblasts. Fission is a more efficient way than de novo assembly to generate new podosome rosettes in these cells. Podosome rosettes undergoing fission possess higher motility and a stronger matrix-degrading capability. Podosome rosette fission may be the result of polarized myosin II-mediated contractility of these structures, which is coordinately regulated by myosin light chain kinase and Rho-associated kinase II. Collectively, this study unveils a previously unknown mechanism-fission for the biogenesis of podosome rosettes.