Poly(ε-caprolactone) (PCL) substrates were modified with thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) brushes to direct and control cellular attachment and detachment. Prior to brush growth, the surface of PCL was activated by a diamine to allow for initiator coupling. Infrared spectra taken before and after cell culturing demonstrated the covalently attached nature of the PNIPAM brushes. PCL is a biocompatible polymer and to prove that the modifications described above did not change this characteristic property, a cell attachment/detachment study was carried out. The modified substrates showed a lower cell attachment when compared to PCL alone and to PCL films modified with the initiator. The possibility to detach the cells in the form of a sheet was proved using PNIPAM-modified PCL films by lowering the temperature to 25 °C. No relevant detachment was shown by the unmodified or by the initiator modified surfaces. This confirmed that the detachment was temperature dependent and not connected to other factors such as polymer swelling. These functionalized polymeric films can find applications as smart cell culture systems in regenerative medicine applications.