We report a new strategy for the preparation of well-defined and mechanically stable porous nanostructures with tunable porosity. Silica inverse opals, which are known as a model system for a porous periodic nanostructure, were grafted with brushes of the thermoresponsive poly(N-isopropylacrylamide) grown via atom transfer radical polymerization. By tuning the temperature, the swelling state of the brush layer is reversibly altered, and with this we were able to control the overall porosity of the system and, thus, the mobility of small penetrants. Fluorescence correlation spectroscopy, a method combining single molecule sensitivity with small probing volume (<1 μm3), was used to directly monitor and quantify in situ the changes in the penetrants’ mobility.