Thermoresponsive polymer-modified microfibers were prepared through electrospinning of poly(4-vinylbenzyl chloride) (PVBC) and subsequent surface-initiated atom transfer radical polymerization for grafting poly(N-isopropylacrylamide) (PIPAAm). Electrospinning conditions were optimized to produce large-diameter (20μm) PVBC microfibers. The amount of PIPAAm grafted on the microfibers was controlled via the IPAAm monomer concentration. The microfibers exhibited thermally controlled cell separation by selective adhesion of normal human dermal fibroblasts in a mixed cell suspension that also contained human umbilical vein endothelial cells. In addition, adipose-derived stem cells (ADSCs) exhibited thermally modulated cell adhesion and detachment, while adhesion of other ADSC-related cells was low. Thus, ADSCs could be separated from a mixture of adipose tissue-derived cells simply by changing the temperature. Overall, the PIPAAm-modified microfibers are potentially applicable as temperature-modulated cell separation materials.Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm) polymer-modified poly(4-vinylbenzyl chloride) (PVBC) microfibers were prepared via electrospinning of PVBC, followed by surface-initiated ATRP. They formed effective thermally-modulated cell separation materials with large surface areas. Cells adhered and extended along the modified microfibers; this was not observed on previously reported PIPAAm-modified flat substrates. The cellular adhesion enabled separation of fibroblast cells, as well as that of adipose-derived mesenchymal stem cells, from mixtures of similar cells. Thus, the temperature-controlled thermoresponsive microfibers would be potentially useful as cell separation materials.