Thermoresponsive poly(benzyl methacrylate)-b-poly(N-isopropylacrylamide) (PBzMA-b-PIPAAm) block co-polymer brush surfaces were prepared by surface-initiated two-step reversible addition-fragmentation chain transfer radical (RAFT) polymerization. PBzMA brushes were fabricated on azoinitiator-immobilized glass substrates in the presence of dithiobenzoate (DTB) compound as a RAFT agent. The amount of grafted polymer was regulated by initial monomer concentrations. The second thermoresponsive blocks were added to the RAFT-related DTB groups located at PBzMA termini through the propagation of PIPAAm chains, resulting in formation of PBzMA-b-PIPAAm brushes. Surface characteristics of the block co-polymer brushes and its influence on thermally regulated cellular behavior were investigated using bovine carotid artery endothelial cells (BAECs), compared with PIPAAm brush surfaces. Cell adhesion/detachment behavior on thermoresponsive polymer brush surfaces significantly depended on their individual polymer architectures and chemical compositions of grafted polymers. Low-temperature treatment at 20°C, below the phase-transition temperature of PIPAAm, induced the spontaneous detachment of adhering cells from the PBzMA-b-PIPAAm brush surfaces with a higher rate than that from PIPAAm brush surfaces. In addition, the cell-repellent effect of the hydrophobic basal layer successfully accelerated for harvesting BAEC sheets from the block co-polymer brush surfaces. Unique features of thermoresponsive block co-polymer brush architectures can be applied to control cell-adhesion strength for enhancing cell adhesion or accelerating cell detachment.