We have developed thermoresponsive microfibers with improved mechanical properties and enhanced temperature modulated-cell separation. Microfiber substrates were electrospun using poly(4-vinylbenzyl chloride) (PVBC)-poly(n-butyl methacrylate) (PBMA) blend materials in different ratios. Although their diameters were similar to those of the PVBC homofibers, polymer-blend microfibers exhibited excellent mechanical properties including non-brittle softness, owing to PBMA with a low T-g. These polymer-blend microfibers enabled the preparation of thin, dense mats that were superior in the experimental handling of cell separation. Poly(N-isopropylacrylamide) (PIPAAm) brushes were grafted via surface-initiated atom transfer radical polymerization from the initiation sites of PVBC in the polymer-blend microfiber substrates. The microfiber in a 25 : 75 ratio of PVBC : PBMA had a reasonable amount of the initiation sites and superior mechanical properties. The PIPAAm-brushed microfibers of the 25 : 75 blend substrate were capable of temperature-modulation, both in terms of wettability and cell separation. Among the normal human dermal fibroblasts (NHDFs), human umbilical vein endothelial cells (HUVECs), and human skeletal muscle myoblasts (HSMMs), HUVEC cells showed significantly poor adhesion on fibers at 37 degrees C; they were separated from adhered NHDF and HSMM cells in the initial step. Reducing the temperature to 20 degrees C remarkably detached NHDF cells, allowing their separation from HSMM cells. Compared with the PIPAAm-brushed PVBC homopolymer microfibers, these cell-separating functions were enhanced in the thermoresponsive PBMA-rich polymer-blend microfibers, probably ascribed to the properties of PBMA and the moderate density of the PIPAAm-brush. Thus, the developed microfibers could be useful for temperature-modulated cell separation systems.