Podosomes are dynamic adhesion structures found in dendritic cells (DCs) and other cells of the myeloid lineage. We previously showed that prostaglandin E2 (PGE₂), an important proinflammatory mediator produced during DC maturation, induces podosome disassembly within minutes after stimulation. Here, we demonstrate that this response is mediated by cAMP elevation, occurs downstream of Rho kinase and is dependent on myosin II. Whereas PGE₂ stimulation leads to activation of the small GTPase RhoA, decreased levels of Rac1-GTP and Cdc42-GTP are observed. These results show that PGE₂ stimulation leads to activation of the RhoA–Rho-kinase axis to promote actomyosin-based contraction and subsequent podosome dissolution. Because podosome disassembly is accompanied by de novo formation of focal adhesions, we propose that the disassembly/formation of these two different adhesion structures is oppositely regulated by actomyosin contractility and relative activities of RhoA, Rac1 and Cdc42.