The surface of nanopores in colloidal films, assembled from 205 nm silica spheres, was modified with poly(Nisopropylacrylamide), PNIPAAM, brushes using surface-initiated ATRP. The polymer thickness inside nanopores was controlled by the polymerization time. The diffusion through PNIPAAM-modified colloidal films was measured using cyclic voltammetry and studied as a function of temperature and polymer brush thickness. Nanopores modified with a thin PNIPAAM brush exhibited a positive gating behavior, where diffusion rates increased with increasing temperature. Nanopores modified with a thick PNIPAAM layer showed a negative gating behavior where diffusion rates decreased with increasing temperature. The observed temperature response is consistent with two transport mechanisms, one in which molecules diffuse through the nanopores Whose volume increases with increasing temperature as the PNIPAAM brush collapses onto the nanopore surface (positive gating) and the second one where molecules diffuse through the porous PNIPAAM that fills the entire nanopore opening and collapses onto itself, becoming hydrophobic and impermeable (negative gating).