Podosomes are actin-rich adhesion and invasion structures. Especially in macrophages, podosomes exist in two subpopulations, large precursors at the cell periphery and smaller podosomes (successors) in the cell interior. To date, the mechanisms that differentially regulate these subpopulations are largely unknown. Here, we show that the membrane-associated protein supervillin localizes preferentially to successor podosomes and becomes enriched at precursors immediately prior to their dissolution. Consistently, podosome numbers are inversely correlated with supervillin protein levels. Using deletion constructs, we find that the myosin II-regulatory N-terminus of supervillin (SV 1-174) is crucial for these effects. Phosphorylated myosin light chain (pMLC) localizes at supervillin-positive podosomes, and time-lapse analyses show that enrichment of GFP-supervillin at podosomes coincides with their coupling to contractile myosin IIA-positive cables. We also show that supervillin binds only to activated myosin IIA, and a dysregulated N-terminal construct (SV 1-830) enhances pMLC levels at podosomes. Thus, preferential recruitment of supervillin to podosome subpopulations may both require and induce actomyosin contractility. Using siRNA and pharmacological inhibition, we demonstrate that supervillin and myosin IIA cooperate to regulate podosome lifetime, podosomal matrix degradation and cell polarization. In sum, we show here that podosome subpopulations differ in their molecular composition and identify supervillin, in cooperation with myosin IIA, as a critical factor in the regulation of podosome turnover and function.