We performed flow-through laboratory experiments on five cylindrically cored samples of ultramafic rocks, in which we generated a well-mated through-going tensile fracture, to investigate evolution of fracture permeability during serpentinization. The samples were tested in a triaxial loading machine at a confining pressure of 50 MPa, pore pressure of 20 MPa, and temperature of 260°C, simulating a depth of 2 km under hydrostatic conditions. A pore pressure difference of up to 2 MPa was imposed across the ends of the sample. Fracture permeability decreased by 1–2 orders of magnitude during the 200–330 h experiments. Electron microprobe and SEM data indicated the formation of needle-shaped crystals of serpentine composition along the walls of the fracture, and chemical analyses of sampled pore fluids were consistent with dissolution of ferro-magnesian minerals. By comparing the difference between fracture permeability and matrix permeability measured on intact samples of the same rock types, we concluded that the contribution of the low matrix permeability to flow is negligible and essentially all of the flow is focused in the tensile fracture. The experimental results suggest that the fracture network in long-lived hydrothermal circulation systems can be sealed rapidly as a result of mineral precipitation, and generation of new permeability resulting from a combination of tectonic and crystallization-induced stresses is required to maintain fluid circulation.